Geert Jannes

Rapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay

SETTING Multidrug resistant Mycobacterium tuberculosis strains are threatening TB control in the world. Rapid diagnosis of resistance is essential for adequate treatment and optimal control of the disease. OBJECTIVE Evaluation of a new technique (Line Probe Assay, LiPA) for easy and rapid detection of Rifampicin resistance (RMPR) of M. tuberculosis. DESIGN After amplification of the region of the RNA polymerase, involved in RMPR, the amplified product is hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip. From the pattern obtained the presence or absence of RMPR M. tuberculosis can be assessed. 67 clinical samples positive in culture for M. tuberculosis were analyzed with LiPA and results were compared with classical susceptibility testing. RESULTS In vitro drug sensitivity testing identified 46 rifampicin sensitive and 21 resistant strains. In 65 of the 67 specimens LiPA results matched classical testing. In two RMPR cases LiPA showed a sensitive pattern. CONCLUSION In contrast to culture and sensitivity testing, where results take on average 6 weeks, LiPA testing is an easy and rapid (< 48 h) method of detecting RMPR M. tuberculosis in clinical samples. Results correlated in 97% of the samples. In the two RMPR samples with a sensitive LiPA pattern another mechanism of resistance is suspected.

Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic separation and PCR.

The rapid detection of an average of 5.9 stressed Salmonella cells in 25 g of food product using immunomagnetic separation (IMS) and PCR is described. For pasteurised egg yolk, egg yolk powder, ice-cream, whole egg, egg white and cheeses made from pasteurised milk PCR was applied after 16 h of preenrichment in buffered peptone water (BPW) using IMS and alkaline lysis as sample preparation method. For whole egg and egg white the BPW was supplemented with iron. For milk powder, and raw milk cheeses, the 16-h preenrichment in BPW was followed by IMS and a 4-h enrichment in Rappaport-Vassiliadis broth. In the latter case, PCR was applied on the enrichment medium after centrifugation and alkaline lysis. For PCR the primers ST11 and ST15 (Aabo et al., 1993) producing a fragment of 429 bp were used. An internal PCR control, designed to be co-amplified with the target DNA using the same primers but producing a smaller fragment of 240 bp, was used.

Rapid Identification of Mycobacteria to the Species Level Using INNO-LiPA Mycobacteria, a Reverse Hybridization Assay

ABSTRACT INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification ofMycobacterium species in culture and identifies theMycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacteriumspecies: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with theMycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species- or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA asM. avium (n = 18) or M. intracellulare (n = 7) or as belonging to theM. avium-M. intracellulare-M. scrofulaceum complex (n = 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.

Polyphasic taxonomy leading to the proposal of Moraxella canis sp. nov. for Moraxella catarrhalis-like strains.

The taxonomic position of a group of 16 Moraxella catarrhalis-like strains, isolated mainly from dogs, was examined by using morphological tests, biochemical tests, serology, ribotyping with oligonucleotide probes, polymerase chain reaction typing of the 16S rRNA gene and the 16S-23S rRNA gene spacer region, polyacrylamide gel electrophoresis of total proteins, fatty acid profiles, moles percent G+C, dot spot and in-solution DNA-DNA hybridizations, and DNA-rRNA hybridizations. It was found that these organisms constitute a distinct cluster within the genus Moraxella. Since they differ genotypically as well as phenotypically from previously described Moraxella species, a new species, Moraxella canis, is proposed to accommodate these isolates. The type strain is LMG 11194 (= N7 = CCUG 8415A).

A Review of Current and Future Molecular Diagnostic Tests for Use in the Microbiology Laboratory

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or micro-arrays, while the introduction of closed-tube real-time polymerase chain reaction systems has resulted in the development of rapid microbial diagnostics with a reduced contamination risk. The use of these new molecular technologies is not restricted to detection and identification of microbial pathogens but also can be used for genotyping, allowing one to determine antibiotic resistance or to perform microbial fingerprinting.

Incidence of Listeria spp. and Listeria monocytogenes in Ready-To-Eat Chicken and Turkey Products Determined by Polymerase Chain Reaction and Line Probe Assay Hybridization

The prevelance of Listeria spp. and Listeria monocytogenes in ready-to-eat poultry products was examined. Following 16 or 48 h of enrichment and selective plating, presumptive Listeria colonies were identified using polymerase chain reaction and reverse hybridization on line probe assay strips. Overall, Listeria spp. and Listeria monocytogenes were found in 35.5% and 15.5% of the poultry samples, respectively. The incidence of Listeria spp. was much higher in unpackaged (41.7%) than in prepackaged (11.1%) poultry products.

Design of a 5′ exonuclease-based real-time PCR assay for simultaneous detection of Bacillus licheniformis, members of the ‘B. cereus group’ and B. fumarioli in gelatine

Aims:  The design of a fast, sensitive and specific detection method for Bacillus licheniformis, members of the ‘B. cereus group’ and B. fumarioli in gelatine.

Unidentified Listeria‐like bacteria isolated from cheese

Unidentified Listeria‐like bacteria, which lack only one of the phenotypic characteristics used to confirm Listeria spp., were isolated from cheese during routine analysis for Listeria monocytogenes. The VIDAS Listeria assay and the Listeria specific PCR or DNA probe assays used did not identify these strains as Listeria species. This group of bacteria was studied for its homogeneity using rep‐PCR and PFGE. Sequence analysis of the 16S rRNA gene showed a homology of 94% to established Listeria spp., implicating a closer relationship than that between Listeria spp. and Brochothrix spp.

Detection and genotyping of human papillomavirus by real-time PCR assay

BACKGROUND Diagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients. OBJECTIVE Our study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples. STUDY DESIGN Validation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity. RESULTS Data showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10(3)cells using in-house HPV (6, 11 and 16) viral load assays. CONCLUSION The real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.

DNA probes for Bordetella species and a colorimetric reverse hybridization assay for the detection of Bordetella pertussis

Three oligonucleotide probe sequences were inferred from the 16S ribosomal ribonucleic acid (rRNA) and the 16S-23S rRNA spacer sequences of Bordetella pertussis ATCC 10380. These probes were used in hybridization tests with deoxyribonucleic acid from Bordetella species and other relevant bacterial taxa. A probe from the spacer region hybridized exclusively to the B. pertussis strains tested and not to strains from other species. Using a combination of three probes, B. pertussis, B. parapertussis/B. bronchiseptica and B. avium could be specifically identified and differentiated from other taxa. Differentiation between B. parapertussis and B. bronchiseptica was not possible with the probes used. Using the spacer probe, a colorimetric hybridization assay specific for B. pertussis was developed based on enzymatic amplification of the 16S-23S rRNA spacer and reverse hybridization in microtitre wells. As compared with results using agarose gel electrophoresis, and Southern and dot-spot hybridization with a 32P-labelled probe, this assay proved to be faster and easier to perform and was found to be at least as sensitive and specific.