Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer.

Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution.

Evaluation of different nucleic acid amplification techniques for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from patients with community-acquired pneumonia.

The number of pathogens involved in community-acquired pneumonia, with varying susceptibilities to antimicrobials, is numerous constituting an enormous challenge for diagnostic microbiology. Differentiation of infections due to Streptococcus pneumoniae and those due to Mycoplasma pneumoniae, Chlamydophila pneumoniae, or L. pneumophila as well as those due to viruses is essential to allow correct decisions concerning the antibiotics to be administered.

Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens.

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.

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens.

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.

Development of conventional and real-time nucleic acid sequence-based amplification assays for detection of Chlamydophila pneumoniae in respiratory specimens.

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.

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.

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.

Sensitivity of detection of rhinoviruses in spiked clinical samples by nucleic acid sequence-based amplification in the presence of an internal control.

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.

Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR.

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.

Development, technical performance, and clinical evaluation of a NucliSens basic kit application for detection of enterovirus RNA in cerebrospinal fluid.

The combination of nucleic acid sequence-based amplification and electrochemiluminescence detection was used to develop an internally controlled, highly sensitive and specific assay for the detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF). The analytical performance of the assay was determined using both in vitro-transcribed EV RNAs and viral culture isolates. The sensitivity of the assay was 10 EV RNA copies per amplification reaction.

Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum.

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.

Onset and duration of cytomegalovirus immediate early 1 mRNA expression in the blood of renal transplant recipients.

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.

Detection of Mycoplasma pneumoniae by real-time nucleic acid sequence-based amplification.

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.

Comparison and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae.

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.

Development and clinical evaluation of an internally controlled, single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species.

A multiplex real-time PCR assay for detection of Legionella pneumophila and Legionella spp. and including an internal control was designed. Legionella species, L.

Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5' noncoding regions of additional rhinovirus strains.

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5' noncoding region (5' NCR) of the viral genome. The nucleotide sequence of the 5' NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses.

Application of NucliSens Basic Kit for the detection of Mycoplasma pneumoniae in respiratory specimens.

A commercially available nucleic acid sequence-based amplification (NASBA) NucliSens Basic Kit (NBK) assay for the detection of Mycoplasma pneumoniae 16S rRNA in respiratory specimens was developed and compared to standard NASBA and PCR assays previously developed in our laboratory. The specificity and sensitivity of the NBK assay was comparable to the specificity and sensitivity of the corresponding standard NASBA assay. The NBK offers standardized reagents for the development of a NASBA assay for the detection of M.

Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed.

Characteristics and applications of nucleic acid sequence-based amplification (NASBA).

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 (>1,000-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.

Quantitative competitive NASBA for measuring mRNA expression levels of the immediate early 1, late pp67, and immune evasion genes US3, US6 and US11 in cells infected with human cytomegalovirus.

Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes.

Sustained virological response in chronic hepatitis C patients after a 6- and a 36-month interferon-alpha2b treatment schedule: a multicenter, randomized, controlled study.

Background: In patients with chronic hepatitis C (HCV) Interferon-alpha (IFN) treatment for 12-18 months is more effective than 6 months in inducing a sustained virological response.

Methods: In a multicenter, randomized, controlled trial, 88 patients with chronic HCV were enrolled (47 treated with IFN-alpha2b and 41 constituted an untreated control group). Treatment consisted of 5 million units (MU) IFN thrice a week (tiw) for 8 weeks and subsequently 2.

Analytical performance and clinical utility of a nucleic acid sequence-based amplification assay for detection of cytomegalovirus infection.

A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants.

Diagnostic value of nucleic-acid-sequence- based amplification for the detection of cytomegalovirus infection in renal and liver transplant recipients.

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.

Characterization of the quantitative HCV NASBA assay.

A quantitative nucleic acid sequence-based amplification assay (NASBA-QT) for detection of hepatitis C virus RNA (HCV-RNA) was evaluated and compared with the HCV branched-DNA (bDNA) assay (Chiron Corporation) and the HCV MONITOR assay (Roche Diagnostic Systems). For this evaluation five panels were designed: (1) serial dilutions of genotype 1b in-vitro HCV-RNA; (2) standards of in-vitro HCV-RNA genotypes 1a, 1b, 2, 3, 4, and 5; (3) a proficiency panel consisting of 12 HCV-RNA positive plasma samples of different genotypes and HCV-RNA concentrations and a genotype 1a and 1b 3-fold dilution series; (4) a panel of 67 HCV-RNA positive plasma samples obtained from patients with HCV infection and (5) an HCV-RNA positive control sample, diluted 50-fold in 25 different HCV-RNA negative plasma samples. The quantitative detection limit was found to be 10(3) copies per 100 microl and the qualitative detection limit 10(2.