24 results on '"Peter Sillekens"'
Search Results
2. Sensitivity of detection of rhinoviruses in spiked clinical samples by nucleic acid sequence-based amplification in the presence of an internal control
- Author
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Margareta Ieven, Katherine Loens, Peter Sillekens, Herman Goossens, and S. R. Pattyn
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Microbiology (medical) ,Serotype ,Rhinovirus ,Nucleic acid sequence based amplification ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Microbiology ,law.invention ,law ,parasitic diseases ,medicine ,Humans ,Viral rna ,Molecular Biology ,Polymerase chain reaction ,Picornaviridae Infections ,Sputum ,Nucleic acid sequence ,RNA ,NASBA ,Molecular biology ,Pharynx ,RNA, Viral ,5' Untranslated Regions ,Bronchoalveolar Lavage Fluid ,Nucleic Acid Amplification Techniques - Abstract
The objectives of the study were to determine the sensitivity of Nucleic Acid Sequence-Based Amplification (NASBA) for the detection of rhinovirus (RV) serotype 15 in water and in spiked respiratory specimens in the presence of a newly developed internal control (IC). The sensitivity of NASBA on RNA was 10 molecules per reaction. The sensitivity of RV NASBA on RV-15 in water and in respiratory specimens was equal to that in tissue culture. Addition of 10(4) molecules of rhinovirus internal control (RV IC) did not affect the sensitivity. Viral RNA should be extracted from clinical specimens as rapidly as possible after collection, since loss of detectable RNA occurs after 2 h. NASBA allows a sensitive detection of RV RNA in spiked respiratory specimens in the presence of an internal control.
- Published
- 2006
3. Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of Chlamydophila pneumoniae in Respiratory Specimens
- Author
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Margaretha Ieven, D. Ursi, T. Beck, Herman Goossens, Katherine Loens, M. Overdijk, and Peter Sillekens
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DNA, Bacterial ,Microbiology (medical) ,Chlamydiology and Rickettsiology ,Respiratory System ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,Sensitivity and Specificity ,law.invention ,Molecular beacon ,law ,RNA, Ribosomal, 16S ,parasitic diseases ,medicine ,Humans ,Self-Sustained Sequence Replication ,Polymerase chain reaction ,Nucleic acid sequence ,Chlamydophila pneumoniae ,Ribosomal RNA ,Virology ,Molecular biology ,NASBA ,Nucleic acid - Abstract
Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae . In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 μl sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.
- Published
- 2006
4. Detection of Rhinoviruses by Tissue Culture and Two Independent Amplification Techniques, Nucleic Acid Sequence-Based Amplification and Reverse Transcription-PCR, in Children with Acute Respiratory Infections during a Winter Season
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P. Oudshoorn, Margareta Ieven, S. Pattyn, C. de Laat, Katherine Loens, Herman Goossens, Peter Sillekens, and H. Foolen
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Microbiology (medical) ,Adolescent ,Rhinovirus ,viruses ,Biology ,medicine.disease_cause ,Sensitivity and Specificity ,Virus ,Microbiology ,Tissue Culture Techniques ,Virology ,parasitic diseases ,Enterovirus Infections ,medicine ,Humans ,Child ,Respiratory Tract Infections ,Self-Sustained Sequence Replication ,Respiratory tract infections ,Reverse Transcriptase Polymerase Chain Reaction ,Viral culture ,Infant, Newborn ,Infant ,Sequence Analysis, DNA ,Nucleic acid amplification technique ,NASBA ,Reverse transcription polymerase chain reaction ,Nasopharyngeal Diseases ,Herpes simplex virus ,Child, Preschool ,Acute Disease ,RNA, Viral ,Reagent Kits, Diagnostic ,Seasons ,Nucleic Acid Amplification Techniques - Abstract
Five hundred seventeen consecutive nasopharyngeal aspirates were collected between October 1998 and May 1999 for episodes of acute respiratory tract infections in children presenting at the University Hospital of Antwerp. Culture and nucleic acid amplification techniques—nucleic acid sequence-based amplification (NASBA) and reverse transcription-PCR (RT-PCR)—were applied to detect rhinoviruses (RVs). Other respiratory viruses were detected by immunofluorescence (IF) analysis of the specimens and IF analysis of shell vial cultures. Among the 517 specimens, 219 viral agents were identified. They were, in decreasing order, rhinoviruses (93 [18.0%]), respiratory syncytial virus (76 [14.7%]), adenoviruses (16 [3.1%]), influenza viruses (15 [2.9%]), enteroviruses (15 [2.9%]), and herpes simplex virus (4 [0.8%]). For the evaluation of rhinovirus detection, culture positivity and/or a positive reaction in the two independent amplification methods was used as an expanded “gold standard.” Based on this standard, the sensitivity, specificity, positive predictive value, and negative predictive value of culture were 44.7, 100, 100, and 99.8%, and those of NASBA and RT-PCR were 85.1, 98.3, 83.3, and 98.5% and 82.9, 93.4, 55.7, and 98.2%, respectively. NASBA and RT-PCR produced comparable results and were significantly more sensitive than virus culture. RVs showed the highest incidence in acute respiratory tract infections in children.
- Published
- 2006
5. Real‐time NASBA detection of SARS‐associated coronavirus and comparison with real‐time reverse transcription‐PCR
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Peter Sillekens, Maria-Cristina Keightley, Charles R. Rinaldo, Kirsten St. George, and Wim Schippers
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RT‐PCR ,viruses ,coronavirus ,medicine.disease_cause ,Severe Acute Respiratory Syndrome ,Article ,NASBA ,Virology ,medicine ,Coronaviridae ,Humans ,skin and connective tissue diseases ,Polymerase ,Coronavirus ,SARS ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,fungi ,RNA ,biology.organism_classification ,Reverse transcription polymerase chain reaction ,Infectious Diseases ,Real-time polymerase chain reaction ,Severe acute respiratory syndrome-related coronavirus ,biology.protein ,RNA, Viral ,Primer (molecular biology) ,Nucleic Acid Amplification Techniques ,Research Article - Abstract
Severe acute respiratory syndrome (SARS) exhibits a high mortality rate and the potential for rapid epidemic spread. Additionally, it has a poorly defined clinical presentation, and no known treatment or prevention methods. Collectively, these factors underscore the need for early diagnosis. Molecular tests have been developed to detect SARS coronavirus (SARS‐CoV) RNA using real time reverse transcription polymerase chain reaction (RT‐PCR) with varying levels of sensitivity. However, RNA amplification methods have been demonstrated to be more sensitive for the detection of some RNA viruses. We therefore developed a real‐time nucleic acid sequence‐based amplification (NASBA) test for SARS‐CoV. A number of primer/beacon sets were designed to target different regions of the SARS‐CoV genome, and were tested for sensitivity and specificity. The performance of the assays was compared with RT‐PCR assays. A multi‐target real‐time NASBA application was developed for detection of SARS‐CoV polymerase (Pol) and nucleocapsid (N) genes. The N targets were found to be consistently more sensitive than the Pol targets, and the real‐time NASBA assay demonstrates equivalent sensitivity when compared to testing by real‐time RT‐PCR. A multi‐target real‐time NASBA assay has been successfully developed for the sensitive detection of SARS‐CoV. J. Med. Virol. 77:602–608, 2005. © 2005 Wiley‐Liss, inc.
- Published
- 2005
6. Onset and duration of cytomegalovirus immediate early 1 mRNA expression in the blood of renal transplant recipients
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Angéla Lukácsi, Valère J. Goossens, Johannes P. van Hooff, Peter Terporten, Cathrien A. Bruggeman, Maarten H. L. Christiaans, Marinus J. Blok, and Peter Sillekens
- Subjects
Adult ,Male ,Human cytomegalovirus ,Time Factors ,Prednisolone ,viruses ,Urinary system ,Cytomegalovirus ,medicine.disease_cause ,Herpesviridae ,Virus ,Immediate-Early Proteins ,Viral Matrix Proteins ,Viral Proteins ,Viral Envelope Proteins ,Betaherpesvirinae ,Virology ,medicine ,Humans ,RNA, Messenger ,Glucocorticoids ,Self-Sustained Sequence Replication ,Aged ,Kidney ,biology ,Age Factors ,virus diseases ,Middle Aged ,Phosphoproteins ,biology.organism_classification ,medicine.disease ,Kidney Transplantation ,Transplantation ,Infectious Diseases ,medicine.anatomical_structure ,Cytomegalovirus Infections ,Immunology ,RNA, Viral ,Female ,medicine.drug - Abstract
Human cytomegalovirus (CMV) messenger (m) RNA expression in circulating leukocytes reflects directly viral activity in the human host. In this study, sixty-nine patients were monitored prospectively for CMV infection and mRNA expression during the first year after renal transplantation. Of the 69 recipients, 58 (84%) recipients were positive for CMV immediate early 1 (IE1) mRNA as detected by nucleic acid sequence-based amplification. The median onset of IE1 expression started at day 22 after transplantation and continued for a median duration of 82 days. IE1 mRNA expression started significantly earlier in recipients who developed an active CMV infection (P = 0.001) and in mycophenolate mofetil (MMF) treated recipients (P = 0.002). The duration of IE1 mRNA expression was significantly longer in recipients that had previously an early onset of IE1 mRNA expression (P = 0.001) and in recipients with active CMV infection (P = 0.007). Remarkably, longer prednisolone intake was correlated with a significantly (P = 0.02) shorter duration of IE1 expression compared to a longer duration of IE1 expression in recipients with only a short prednisolone intake. In recipients infected with glycoprotein B (gB) type 1 CMV strains, the duration of IE1 expression was significantly (P = 0.04) shorter compared to recipients infected with non-gB type 1 CMV strains (64 days vs. 150 days). The study indicates that multiple factors play a role in the onset and/or duration of CMV IE1 mRNA expression, for example, MMF treatment, prednisolone intake, and gB type of the specific CMV strain. The clinical significance of these correlations remains to be studied in more detail. J. Med. Virol. 72:94–101, 2004. © 2004 Wiley-Liss, Inc.
- Published
- 2003
7. Development and clinical evaluation of an internally controlled, single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species
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Kate Templeton, Herman Goossens, Sitha A. Scheltinga, Jantine W. Crielaard, Peter Sillekens, Eric C. J. Claas, Alje P. van Dam, and Academic Medical Center
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Microbiology (medical) ,biology ,Legionella ,Reproducibility of Results ,Bacteriology ,biology.organism_classification ,bacterial infections and mycoses ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Legionella pneumophila ,Virology ,Microbiology ,respiratory tract diseases ,Single tube ,Real-time polymerase chain reaction ,Multiplex polymerase chain reaction ,Humans ,bacteria ,Multiplex ,Human medicine ,Legionella species ,Clinical evaluation - Abstract
A multiplex real-time PCR assay for detection of Legionella pneumophila and Legionella spp. and including an internal control was designed. Legionella species, L. pneumophila , and the internal control were detected simultaneously by probes labeled with 6-carboxy-fluorescein, hexachlorofluorescein, and indodicarbocyanine, respectively. Therefore, no postamplification analysis was required in order to distinguish the targets. The sensitivity of both assays was 2.5 CFU/ml, and from analysis of 10 culture-positive and 74 culture-negative samples from patients investigated for legionellosis, 100% agreement was observed by both assays in comparison to culture. Four additional positives were found by the multiplex real-time PCR assay in the Legionella culture-negative samples.
- Published
- 2003
8. EARLY DETECTION OF HUMAN CYTOMEGALOVIRUS INFECTION AFTER KIDNEY TRANSPLANTATION BY NUCLEIC ACID SEQUENCE-BASED AMPLIFICATION1
- Author
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Valère J. Goossens, Marinus J. Blok, M. H. L. Christiaans, Cathrien A. Bruggeman, J.P. van Hooff, Peter Sillekens, and Jaap M. Middeldorp
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Human cytomegalovirus ,Transplantation ,Congenital cytomegalovirus infection ,Biology ,medicine.disease_cause ,medicine.disease ,biology.organism_classification ,Virology ,NASBA ,Herpesviridae ,Immediate early protein ,Serology ,Betaherpesvirinae ,parasitic diseases ,Immunology ,medicine - Abstract
Background. The early detection of human cytomegalovirus infection after organ transplantation is a prerequisite for effective antiviral therapy. We evaluated the diagnostic value of monitoring the viral immediate-early (IE) 1 mRNA expression in blood leukocytes by nucleic acid sequence-based amplification (NASBA). Methods. Nucleic acids were isolated from 489 blood samples collected from 42 kidney transplant recipients and subjected to amplification by IE NASBA The IE NASBA results were compared to those from pp67 NASBA, pp65 antigenemia, cell culture (DEAFF and CPE), and serology. Results. IE NASBA provedl to be the most sensitive assay which detected the onset of both primary and secondary cytomegalovirus infection significantly earlier than the other assays. Conclusions. The early detection of cytomegalovirus infection with IE NASBA would enable the start of effective antiviral therapy at an early state of infection to prevent cytomegalovirus disease in patients at risk.
- Published
- 1999
9. Diagnostic value of nucleic-acid-sequence- based amplification for the detection of cytomegalovirus infection in renal and liver transplant recipients
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J.P. van Hooff, Valère J. Goossens, Krister Höckerstedt, Peter Sillekens, Marinus J. Blok, Irmeli Lautenschlager, Jaap M. Middeldorp, Cathrien A. Bruggeman, M. H. L. Christiaans, Pathology, CCA - Cancer immunology, CCA - Target Discovery & Preclinial Therapy Development, CCA - Biomarkers, CCA - Evaluation of Cancer Care, AII - Infectious diseases, and AII - Cancer immunology
- Subjects
Virus Cultivation ,Genes, Viral ,medicine.medical_treatment ,viruses ,Congenital cytomegalovirus infection ,Cytomegalovirus ,Liver transplantation ,Polymerase Chain Reaction ,Immediate early protein ,Serology ,Immediate-Early Proteins ,Viral Matrix Proteins ,Postoperative Complications ,Virology ,parasitic diseases ,medicine ,Humans ,RNA, Messenger ,Antigens, Viral ,Genes, Immediate-Early ,Kidney transplantation ,business.industry ,Viral culture ,virus diseases ,Nucleic acid amplification technique ,medicine.disease ,Phosphoproteins ,NASBA ,Kidney Transplantation ,Liver Transplantation ,Infectious Diseases ,surgical procedures, operative ,Immunology ,Cytomegalovirus Infections ,RNA, Viral ,business ,Nucleic Acid Amplification Techniques - Abstract
To evaluate the diagnostic value of nucleic-acid-sequence-based amplification (NASBA) for the detection of cytomegalovirus (CMV) infection in transplant recipients, we compared immediate early 1 (IE1) and late pp67 mRNA detection by NASBA with the antigenemia assay, PCR and viral culture in 72 renal transplant (RTx) recipients and with antigenemia and serology in 25 liver transplant (LTx) recipients. Antigenemia, viral culture and pp67 NASBA were almost equivalent for the detection of CMV in RTx recipients. In LTx recipients, antigenemia detected more positive samples and more positive recipients compared to pp67 NASBA. In RTx recipients, PCR detected more positive samples and positive recipients compared to pp67 NASBA, antigenemia and viral culture. Also the first day of detection was slightly earlier for PCR. However, IE1 NASBA was the most sensitive test and detected 96% of all positive samples and positive transplant recipients. In addition, IE1 NASBA preceded PCR and all other positive results. This makes IE1 NASBA a very attractive screening test for the early detection of CMV infection.
- Published
- 1999
10. 2181 A solution for same-day extended RAS testing
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Ina Vandenbroucke, Geert Maertens, M. Van Brussel, Peter Sillekens, Erwin Sablon, and E. Bellon
- Subjects
Cancer Research ,Oncology - Published
- 2015
11. Serological and molecular analysis of hepatitis C virus envelope regions 1 and 2 during acute and chronic infections in chimpanzees
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Fons Bosman, Guy de Martinoff, Kitty van Hoek, Geert Maertens, Ton Kos, Peter Sillekens, Wim Quint, Inge Frantzen, Lieven Stuyver, and Leen-Jan van Doorn
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Male ,Pan troglodytes ,Hepatitis C virus ,Viremia ,Hepacivirus ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,Virus ,Serology ,Evolution, Molecular ,Viral Envelope Proteins ,Viral envelope ,Virology ,medicine ,Animals ,Cloning, Molecular ,Phylogeny ,DNA Primers ,Base Sequence ,medicine.disease ,Hepatitis C ,Chronic infection ,Infectious Diseases ,Acute Disease ,Chronic Disease ,Immunology ,RNA, Viral ,Female ,Viral disease ,Viral load - Abstract
Acute and chronic Hepatitis C virus infections were investigated retrospectively in chimpanzees that had been infected from a single source. Anti-E1 and anti-E2 were detected in two of three chimpanzees with a chronic infection, but were first detected 1 to 2 years after inoculation. Sequence evolution of the E1 region in three animals over a period of 9 to 11 years revealed a mutation rate of 1.02 to 2.23 × 10 base substitutions per site per year. The acute phase viremia levels in acute infections which resolved appeared to be at least 10-fold higher than during the acute phase of chronic infections. During chronic infections, the viral load fell rapidly after the acute phase and remained at very low levels for several years. After 4 to 6 years, the viral load and liver enzymes increased again, suggesting reactivation of the infection. There was no clear temporal relationship between sequence evolution of the E1 region, changes in viral load, and the production of antibodies to the envelope proteins. J. Med. Virol. 52:441–450, 1997. © 1997 Wiley-Liss, Inc.
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- 1997
12. Serum HCV RNA levels assessed by quantitative NASBA®: stability of viral load over time, and lack of correlation with liver disease
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William L. Irving, Keith R. Neal, Peter Sillekens, Rosalind C. Hollingsworth, and Peter van Deursen
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Hepatology ,biology ,Hepacivirus ,Hepatitis C virus ,Hepatitis C ,Nucleic acid amplification technique ,medicine.disease ,biology.organism_classification ,medicine.disease_cause ,Virology ,Virus ,Liver disease ,medicine ,Viral disease ,Viral load - Abstract
Background/Aims: We used the hepatitis C virus quantitative NASBA® technique to evaluate the stability of viral load within individuals with chronic hepatitis C, to determine the range of viraemic load between individuals, and to assess the usefulness of hepatitis C virus RNA quantitation in predicting the severity of underlying hepatitis C virus-induced liver disease. Methods: Hepatitis C virus RNA was determined, using the quantitative NASBA® assay, in multiple serum samples from 11 individuals with chronic hepatitis C over an average time period of 11 months (range=3–23 months), and in single serum samples from a further 10 individuals. Results: In 10/11 individuals the hepatitis C virus RNA titres were within one log 10 copies/ml of each other during this time period. In the eleventh, there was a rise of 1.36 log 10 copies/ml in two serum samples taken 8 months apart. The viraemic load varied by 2.79 log 10 copies/ml serum between individuals. There were no correlations between mean RNA levels and total biopsy scores (either Knodell or Sheffield scores), or the individual components of the biopsy scoring systems, except the sinusoidal infiltration component of the Sheffield score. There was also no difference in viral RNA levels between those infected with type 1 as compared to type 3 virus, with a mean level in both groups of 7.2 log 10 copies/ml. Conclusions: Hepatitis C virus serum RNA level is stable within individuals within the studied time period. Viral load varies between infected individuals but is not a useful prognostic indicator of the severity of virus-induced liver disease.
- Published
- 1996
13. Detection of Mycoplasma pneumoniae by Real-Time Nucleic Acid Sequence-Based Amplification
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D. Ursi, Katherine Loens, Margaretha Ieven, Peter Sillekens, M. Overdijk, Herman Goossens, and T. Beck
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Microbiology (medical) ,Mycoplasma pneumoniae ,Nucleic acid sequence ,RNA ,Bacteriology ,Mycoplasmataceae ,Ribosomal RNA ,Biology ,medicine.disease_cause ,biology.organism_classification ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Virology ,NASBA ,respiratory tract diseases ,Microbiology ,RNA, Ribosomal, 16S ,parasitic diseases ,medicine ,Nucleic acid ,Humans ,Sputum ,medicine.symptom ,Self-Sustained Sequence Replication - Abstract
Real-time isothermal nucleic acid sequence-based amplification (RT-NASBA) was applied to the detection of Mycoplasma pneumoniae . In vitro-generated M. pneumoniae RNA was used to assess the sensitivity of the assay. The 95% hit rate was 148 molecules of M. pneumoniae RNA in the amplification and 10 4 molecules of in vitro-generated RNA after nucleic acid extraction. The sensitivity of the RT-NASBA and the conventional NASBA assays corresponded to 5 color-changing units (CCU) of M. pneumoniae . In spiked throat swabs, nasopharyngeal aspirates, bronchoalveolar lavages, and sputum, the sensitivity of both NASBA assays corresponded to 5 to 50 CCU of M. pneumoniae . A total of 17 clinical specimens positive for M. pneumoniae by PCR were also positive by conventional NASBA, but one specimen was negative by RT-NASBA. These results indicate that the sensitivity of detection of M. pneumoniae by RT-NASBA in respiratory samples might be slightly reduced compared to that by conventional NASBA. However, the real-time assay is superior in speed and ease of handling.
- Published
- 2003
14. Immunochemical structure of the carboxy-terminal part of hepatitis B e antigen: identification of internal and surface-exposed sequences
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Vadim Bichko, Pauls Pumpens, Peter Sillekens, Peter Pushko, Elmars Grens, Britta Wahren, Matti Sällberg, Ivar Berzinsh, Michael Noah, and Lars O. Magnius
- Subjects
Protein Denaturation ,Antigenicity ,medicine.drug_class ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Biology ,Monoclonal antibody ,Epitope ,Epitopes ,Antigen ,Antibody Specificity ,Virology ,medicine ,Humans ,Amino Acid Sequence ,Hepatitis B e Antigens ,Antibodies, Monoclonal ,virus diseases ,Hepatitis B ,Hepatitis B Core Antigens ,Fusion protein ,digestive system diseases ,HBcAg ,HBeAg ,Immunoglobulin G ,Carrier State ,biology.protein ,Antibody - Abstract
The C-terminal region of hepatitis B virus (HBV) e antigen (HBeAg), amino acids (aa) 121 to 147, was characterized for reactivity with 15 monoclonal antibodies (MAbs) and sera from 16 chronic carriers on the HB surface antigen with anti-HBe. Recombinant proteins exposing fragments of the HBc/e polypeptide (aa 29 to 176, 60 to 176, 101 to 176, 121 to 176, 134 to 176, 138 to 176, 139 to 176, 140 to 176, 146 to 176 and 156 to 176) fused to the N terminus of the coat protein of RNA phage fr were constructed, as were two sets of synthetic peptides covering residues 121 to 136 and 130 to 147, where each residue was sequentially substituted by alanine. The MAbs were found to recognize overlapping epitopes in the fusion proteins within residues 121 to 176; however, none of the MAbs reacted with proteins covering residues 146 to 176 and 156 to 176. Using the synthetic peptides it was found that the MAbs recognized epitopes at residues 128-TPPAYR-133, 133-RPPNAP-138, 135-PNAPIL-140, 138-PILSTLPE-145 and 143-LPET-146. Only MAbs recognizing the epitope 128-TPPAYR-133 were found to react with both native HBeAg and denatured HBcAG, whereas MAbs recognizing epitopes located closer to the C terminus of HBeAg were reactive only with denatured HBcAg. The recognition sites for the human IgG1 overlapped with the epitopes of the MAbs recognizing native HBeAg. Our interpretation of these findings is that the region 124 to 133 is on the surface of native HBeAg and denatured HBcAg, and that the adjacent region 135 to 147 is not accessible on the surface of native HBeAg, but becomes exposed on denatured HBcAg.
- Published
- 1993
15. Development of real-time multiplex nucleic acid sequence-based amplification for detection of **Mycoplasma pneumoniae**, **Chlamydophila pneumoniae**, and **Legionella** spp. in respiratory specimens
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Peter Sillekens, Katherine Loens, T. Beck, M. Overdijk, Margareta Ieven, Herman Goossens, and D. Ursi
- Subjects
Microbiology (medical) ,Mycoplasma pneumoniae ,Legionella ,Bronchi ,Biology ,medicine.disease_cause ,Legionella pneumophila ,Sensitivity and Specificity ,Microbiology ,Molecular beacon ,RNA, Ribosomal, 16S ,Pneumonia, Mycoplasma ,medicine ,Humans ,Multiplex ,Chlamydophila Infections ,Self-Sustained Sequence Replication ,DNA Primers ,Netherlands ,Legionellosis ,Sputum ,Bacteriology ,Chlamydophila pneumoniae ,biology.organism_classification ,Virology ,NASBA ,respiratory tract diseases ,RNA, Bacterial ,Mollicutes ,Pharynx ,Bronchoalveolar Lavage Fluid - Abstract
Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae , and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae , and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae -, C. pneumoniae -, and L. pneumophila -positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae , and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.
- Published
- 2008
16. Evaluation of a sensitive and fully automated extended KRAS test for detection of 21 mutations in 6 codons in KRAS exons 2, 3 and 4
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Mark M. Kockx, Geert Maertens, Peter Sillekens, Ann Torremans, Erwin Sablon, Marianne Van Brussel, Ina Vandenbroucke, Isabelle Vanden Bempt, and E. Bellon
- Subjects
Neuroblastoma RAS viral oncogene homolog ,Genetics ,Cancer Research ,business.industry ,Colorectal cancer ,medicine.disease ,medicine.disease_cause ,digestive system diseases ,Clinical Practice ,Exon ,Oncology ,Fully automated ,Cancer research ,Medicine ,KRAS ,business ,neoplasms - Abstract
e22147 Background: Detection of an extended range of mutations in KRAS and NRAS exons 2, 3 and 4 for colorectal cancer (CRC) tissues is now accepted in clinical practice (Douillard et al. NEJM 2013...
- Published
- 2015
17. Development of conventional and real-time NASBA® for the detection of **Legionella** species in respiratory specimens
- Author
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D. Ursi, Margareta Ieven, M. Overdijk, Katherine Loens, Herman Goossens, Peter Sillekens, and T. Beck
- Subjects
Microbiology (medical) ,Serotype ,Legionella ,Microbiology ,Legionella pneumophila ,Sensitivity and Specificity ,Molecular beacon ,RNA, Ribosomal, 16S ,parasitic diseases ,Bile ,Humans ,Molecular Biology ,Lung ,Self-Sustained Sequence Replication ,Legionellosis ,biology ,Sputum ,Reproducibility of Results ,bacterial infections and mycoses ,biology.organism_classification ,16S ribosomal RNA ,Virology ,NASBA ,respiratory tract diseases ,Body Fluids ,RNA, Bacterial ,Luminescent Measurements ,Nucleic acid ,bacteria ,Legionnaires' Disease ,Bronchoalveolar Lavage Fluid ,Bacteria - Abstract
Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.
- Published
- 2006
18. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum
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Gerard J. Schoone, Robert W. Sauerwein, Peter Sillekens, Petra Schneider, Rob Hermsen, Liselotte Wolters, Henk D. F. H. Schallig, KIT: Biomedical Research, and Medical Microbiology and Infection Prevention
- Subjects
Microbiology (medical) ,Plasmodium falciparum ,Population ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Auto-immunity, transplantation and immunotherapy [N4i 4] ,Molecular beacon ,parasitic diseases ,Animals ,Humans ,Multiplex ,Malaria, Falciparum ,education ,Self-Sustained Sequence Replication ,education.field_of_study ,biology ,Poverty-related infectious diseases [N4i 3] ,Reproducibility of Results ,DNA, Protozoan ,biology.organism_classification ,DNA extraction ,Molecular biology ,Real-time polymerase chain reaction ,Nucleic acid ,Parasitology ,RNA extraction ,Microbial pathogenesis and host defense [UMCN 4.1] ,Infection and autoimmunity [NCMLS 1] - Abstract
Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum , such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 μl of venous blood to achieve this sensitivity, compared to 50 μl of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.
- Published
- 2005
19. Comparison and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae
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Matthias F. C. Beersma, Sitha A. Scheltinga, Herman Goossens, Eric C. J. Claas, A Willy Graffelman, Kate Templeton, Peterhans J. van den Broek, Jolanda M. van Schie, Jantine W. Crielaard, and Peter Sillekens
- Subjects
Microbiology (medical) ,Adult ,Male ,Mycoplasma pneumoniae ,Mycoplasmataceae ,medicine.disease_cause ,Polymerase Chain Reaction ,Sensitivity and Specificity ,law.invention ,Serology ,Microbiology ,law ,Pneumonia, Mycoplasma ,medicine ,Humans ,Serologic Tests ,Polymerase chain reaction ,Aged ,biology ,Bacteriology ,Nucleic acid amplification technique ,Middle Aged ,biology.organism_classification ,Virology ,NASBA ,Real-time polymerase chain reaction ,Mollicutes ,Female ,Human medicine ,Nucleic Acid Amplification Techniques - Abstract
Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasma -positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae , providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.
- Published
- 2003
20. Characteristics and applications of nucleic acid sequence-based amplification (NASBA)
- Author
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Birgit Deiman, Pierre van Aarle, and Peter Sillekens
- Subjects
Models, Molecular ,Nucleic acid sequence ,Recombinase Polymerase Amplification ,RNA ,Bioengineering ,DNA ,Amplicon ,Biology ,Applied Microbiology and Biotechnology ,Biochemistry ,Molecular biology ,NASBA ,chemistry.chemical_compound ,chemistry ,Molecular beacon ,parasitic diseases ,Nucleic acid ,Humans ,Molecular Biology ,Self-Sustained Sequence Replication ,Biotechnology - Abstract
Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. In some NASBA systems, DNA is also amplified though very inefficiently and only in the absence of the corresponding RNA target or in case of an excess (>1000-fold) of target DNA over RNA. As NASBA is primer-dependent and amplicon detection is based on probe binding, primer and probe design rules are included. An overview of various target nucleic acids that have been amplified successfully using NASBA is presented. For the isolation of nucleic acids prior to NASBA, the “Boom” method, based on the denaturing properties of guanidine isothiocyanate and binding of nucleic acid to silica particles, is preferred. Currently, electro-chemiluminescence (ECL) is recommended for the detection of the amplicon at the end of amplification. In the near future, molecular beacons will be introduced enabling “real-time detection,” i.e., amplicon detection during amplification. Quantitative HIV-1 NASBA and detection of up to 48 samples can then be performed in only 90 min.
- Published
- 2002
21. Bovine immunodeficiency virus: immunochemical characterization and serological survey
- Author
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Marian C. Horzinek, Marck J. M. Koolen, Lian C.E.J.M. Keldermans, John Black, Peter Sillekens, Thea Stuurman, and A. A. P. M. Herrewegh
- Subjects
Diergeneeskunde ,Immunodeficiency Virus, Bovine ,Radioimmunoprecipitation Assay ,medicine.drug_class ,Population ,Blotting, Western ,Cattle Diseases ,Fluorescent Antibody Technique ,Enzyme-Linked Immunosorbent Assay ,Monoclonal antibody ,Virus ,Serology ,Western blot ,Virology ,medicine ,Animals ,Serologic Tests ,education ,Netherlands ,education.field_of_study ,biology ,medicine.diagnostic_test ,Bovine immunodeficiency virus ,Antibodies, Monoclonal ,RNA-Directed DNA Polymerase ,biology.organism_classification ,Blot ,biology.protein ,Lentivirus Infections ,Cattle ,Antibody - Abstract
Bovine immunodeficiency virus (BIV) was purified by isodensity centrifugation; viral activities were monitored in gradient fractions using the reverse transcriptase assay and a p26-specific monoclonal antibody ELISA. In the coincident peak fractions (density about 1.17 g/ml) proteins with M r values of 26K, 17K, 53K, 14K and 100K (with decreasing intensity) were detected by Western blotting using serum of a calf after experimental BIV infection. When 957 randomly collected cattle sera from The Netherlands were tested by indirect immunofluorescence and confirmed using Western blot and/or radioimmunoprecipitation, 1.4% appeared seropositive. Thus BIV infection is not uncommon in one European cattle population.
- Published
- 1991
22. Contents Vol. 42, 1999
- Author
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Margaritis Kountidis, Petra Reinke, Peter Sillekens, Erik A M Verschuuren, Ralf Ewert, Anita S.-F. Chong, Stephan Stenglein, Martin Scholz, Cathrien A. Bruggeman, Pieter C. Limburg, M. Scholz, Nicholas Davis-Poynter, Martin C. Harmsen, Marinus J. Blok, JiKun Shen, Vera S. Donnenberg, L. van Renterghem, Klaus Kleinschmidt, Klaus Mayr, Banas Richard A, Florian Kern, Wolf-Dietrich Döcke, Detlef Michel, Adri ana M. Kas-Deelen, Jeroen Kloover, Jaap M. Middeldorp, Irmeli Lautenschlager, Peter Schaarschmidt, Kaisa Holma, Willem J. van Son, Gerhard Jahn, Rainer Voisard, Jane Cai, Nell S. Lurain, Boelo Meedendorp, Griffith Bp, James H. Dauber, R. Vanholder, Arie P. van den Berg, Patrick S. Beisser, Hans-Dieter Volk, Cecilia Söderberg-Nauclér, Mamun Ahmed, Robert J. Keenan, M.P. Landini, James W. Williams, K. Höckerstedt, J. Cinatl, Valère J. Goossens, Bianca Vaida, J.-U. Vogel, Ulrich Wenderoth, Adriana Zeevi, Eltjo F. de Maar, P. Spezzacatena, Mariapia A. Degli-Esposti, Jürgen E. Gschwend, Colleen M. Cebulla, Regine Baur, Cathrien Bruggeman, Nicole Faulhaber, L. Gabrielli, Albert Ramon, Daniel M. Miller, Anu Soots, H. Vanpoucke, Anke Lüske, Kaija Inkinen, Helen E. Farrell, Barbara Reinhardt, Christian Sinzger, Vincent C. Emery, Albert D. Donnenberg, Susanne Riegler, H.W. Doerr, Susanna Prösch, Leonard Blinder, Raisa Loginov, Kathy Spichty, Claudia Frömmel, Thomas Mertens, Cornelis Vink, Deborah A. Knight, Wim van der Bij, Aldo Iacono, Hermann Einsele, Daniel D. Sedmak, R.A. Blaheta, Juhani Ahonen, Harri Kauppinen, R. Kotchetkov, W. James Waldman, Johannes P. van Hooff, Tiziana Lazzarotto, Susan Michelson, Jay A. Nelson, Maarten H.L. Christiaans, Detlev H. Krüger, Leena Krogerus, B. van Vlem, S. Varani, and Elham Khatamzas
- Subjects
Infectious Diseases ,Virology - Published
- 1999
23. Subject Index Vol. 42, 1999
- Author
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S. Varani, Mamun Ahmed, Florian Kern, Patrick S. Beisser, Cecilia Söderberg-Nauclér, Erik A M Verschuuren, Peter Schaarschmidt, Willem J. van Son, Nicholas Davis-Poynter, Eltjo F. de Maar, Klaus Mayr, Detlef Michel, Griffith Bp, Adri ana M. Kas-Deelen, Harri Kauppinen, Raisa Loginov, Jay A. Nelson, Maarten H.L. Christiaans, Vincent C. Emery, Susanne Riegler, Ulrich Wenderoth, Daniel M. Miller, Martin Scholz, Klaus Kleinschmidt, Martin C. Harmsen, Wolf-Dietrich Döcke, Kaisa Holma, Marinus J. Blok, Detlev H. Krüger, Stephan Stenglein, Leena Krogerus, Boelo Meedendorp, P. Spezzacatena, B. van Vlem, Elham Khatamzas, M. Scholz, Valère J. Goossens, Vera S. Donnenberg, Hermann Einsele, Nicole Faulhaber, L. Gabrielli, Anu Soots, Aldo Iacono, Peter Sillekens, Anke Lüske, H.W. Doerr, Gerhard Jahn, K. Höckerstedt, Bianca Vaida, Juhani Ahonen, J.-U. Vogel, Susanna Prösch, Anita S.-F. Chong, Tiziana Lazzarotto, Susan Michelson, Daniel D. Sedmak, Robert J. Keenan, W. James Waldman, R. Vanholder, J. Cinatl, M.P. Landini, Johannes P. van Hooff, Cathrien Bruggeman, Jane Cai, Kathy Spichty, R.A. Blaheta, Kaija Inkinen, Mariapia A. Degli-Esposti, Nell S. Lurain, Ralf Ewert, Albert D. Donnenberg, Irmeli Lautenschlager, James H. Dauber, James W. Williams, Albert Ramon, Hans-Dieter Volk, Petra Reinke, Adriana Zeevi, H. Vanpoucke, Helen E. Farrell, Cornelis Vink, Wim van der Bij, JiKun Shen, L. van Renterghem, Pieter C. Limburg, Margaritis Kountidis, Banas Richard A, Jeroen Kloover, Jaap M. Middeldorp, Arie P. van den Berg, Leonard Blinder, Colleen M. Cebulla, Cathrien A. Bruggeman, Barbara Reinhardt, Claudia Frömmel, Deborah A. Knight, Rainer Voisard, Jürgen E. Gschwend, Regine Baur, Christian Sinzger, Thomas Mertens, and R. Kotchetkov
- Subjects
Infectious Diseases ,Index (economics) ,Virology ,Statistics ,Subject (documents) ,Mathematics - Published
- 1999
24. Detection of viral mRNAs as a diagnostic tool for CMV infection in humans
- Author
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Peter Sillekens
- Subjects
Infectious Diseases ,Virology ,Biology - Published
- 1999
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