Technical and clinical validation of three commercial real-time PCR kits for the diagnosis of neuroborreliosis in cerebrospinal fluid on three different real-time PCR platforms.

This study reports the evaluation of the technical and clinical validation of the O-DiaBorburg kit (DIA), Borrelia burgdorferi PCR kit, ISEX (GENE), and Borrelia burgdorferi sensu lato Real-TM (SAC) for the diagnosis of neuroborreliosis in cerebrospinal fluid based on both Borrelia DNA and CSF samples from patients with clinical suspicion of neuroborreliosis. This validation study was done by analysing the kits on the Rotorgene Q (RGQ), CFX96, and LightCycler480 (LC480). For all kits, the linear range was larger on RGQ than on CFX96 and LC480. A good reproducibility was obtained for all assays on all instruments. Storage at -20 °C resulted in a decreased reproducibility for SAC. Results of the limit of detection (LOD95) experiments indicated a better sensitivity than described in the kit insert for all kits on all PCR platforms. No cross-reactivity was found for genetically related organisms nor for other pathogens which may be present in CSF. All species of the Borrelia burgdorferi sensu lato complex were detected with the GENE and SAC kits. The DIA kit failed to detect B. lusitaniae. The results seemed to indicate a better overall performance for the GENE kit on RGQ. However, its diagnostic value could not be confirmed in the clinical validation study, wherein none of the 103 CSF samples from clinical neuroborreliosis cases showed a positive real-time PCR result with the GENE kit analysed on RGQ.

Comparison of different phenotypic assays for the detection of extended-spectrum ß-lactamase production by inducible AmpC-producing Gram-negative bacilli.

Routine detection of extended-spectrum ß-lactamase (ESBL) production by AmpC-producing Enterobacteriaceae in microbiology laboratories is still a problem. The aim of this study was to compare the performance of four different phenotypic ESBL confirmation assays within this group of Enterobacteriaceae. A total of 83 AmpC-inducible Enterobacteriaceae were included in this study (58 clinical isolates with presumptive ESBL production and 25 molecularly characterized ESBL-producing isolates). Each isolate was tested for the presence of an ESBL enzyme by four phenotypic ESBL confirmation assays: ESBL Etests and combined double-disk synergy tests (CDDST), both on Mueller-Hinton (MH) agar with and without the use of cloxacillin, an AmpC inhibitor. Our study showed that performing a CDDST on MH agar with cefotaxime as the only indicator cephalosporin is not a reliable way to detect ESBL-encoding genes among chromosomal AmpC-producing Enterobacteriaceae due to its low sensitivity (52 %). The use of cloxacillin in this CDDST could only significantly increase the specificity of the CDDST when used with ceftazidime as the indicator [sensitivity (SN), 92 %; specificity (SP), 93 %]. Regarding ESBL Etest® strips, the sensitivity of the cefepime strip (80 %) was significantly higher compared to the cefotaxime and ceftazidime strips (16 % and 32 %, respectively). Adding cloxacillin to the MH agar improved the ESBL detection of each of these strips. We recommend the CDDST on MH agar supplemented with cloxacillin and ceftazidime or cefepime as the indicator cephalosporin as the most cost-efficient strategy to confirm ESBL production in inducible AmpC-producing Enterobacteriaceae.

Timely diagnosis of respiratory tract infections: evaluation of the performance of the Respifinder assay compared to the xTAG respiratory viral panel assay.

BACKGROUND: Respiratory tract infections are the most common cause of hospitalization in infants and young children and are typically caused by viral or, less commonly, bacterial pathogens. Existing non-molecular diagnostic methods have several drawbacks such as limited sensitivity, long turn-a-round time and limited number of pathogens that can be detected. OBJECTIVES: Nucleic acid amplification methods can increase sensitivity and enable the initiation of appropriate interventions without delay. Broad-spectrum detection and identification circumvent the use of individual diagnostic DNA or RNA based assays. At present, several commercial assays are available for broad-spectrum detection. STUDY DESIGN: We compared the performance of the xTAG Respiratory Viral Panel (RVP) (Luminex Molecular Diagnostics, Toronto, Canada) with that of the Respifinder (Pathofinder, Maastricht, Netherlands) for 9 external quality assurance (EQA) panels (QCMD, Scotland) consisting of a total of 106 EQA samples. RESULTS: Both the RVP and the Respifinder assay have an excellent specificity. Sensitivity was 33% and 78% for the RVP and the Respifinder assay, respectively. For both assays, sensitivity was low for weak positive samples. DISCUSSION: The results of our study seem to indicate a better sensitivity for the Respifinder. Analysis of patient samples is necessary to evaluate the clinical performance.

Reflections and proposals to assure quality in molecular diagnostics.

Molecular diagnostic testing has become an important tool in clinical laboratories. Accreditation according to the international quality standard ISO15189:2007 for medical laboratories is required for reimbursement of several molecular diagnostic tests in Belgium. Since the ISO15189:2007 standard applies to medical laboratories in general, the particular requirements for quality and competence are mentioned in general terms, not taking into account the specificities of molecular biology testing. Therefore, the working group "MolecularDiagnostics.be" described a consensus interpretation of chapter 5, Technical requirements, of the ISO standard for application in molecular diagnostic laboratories. The manuscript can be used as an instrument to prepare internal and external audits that meet the 15015189:2007 (chapter 5) criteria.