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G Muyldermans

Publications and source records attributed to G Muyldermans.

At least 19 recordsLinked to original sources
PubMed2005

External quality assessment for molecular detection of Bordetella pertussis in European laboratories.

Although the PCR for the detection of Bordetella pertussis is routinely performed in diagnostic laboratories, no quality assessment program has so far been described. We report on the results obtained with two external quality assessment proficiency panels sent to European laboratories. The first proficiency panel contained a series of dilutions of three previously characterized B. pertussis clinical isolates and two negative controls. No false-positive results were reported by six laboratories providing seven data sets. The reported limits of detection of the three B. pertussis strains varied between 4 and 4,000, 9 and 9,000, and 3 and 30,000 CFU/ml, respectively. The second proficiency panel, composed of a series of dilutions of reference strains of B. pertussis, B. holmesii, B. hinzii, and B. bronchiseptica, as well as negative controls, was sent to nine laboratories. One laboratory reported a negative result for a sample and reported a B. parapertussis-positive sample to be positive for B. pertussis. By using the B. pertussis-specific target gene pertactin, one laboratory detected B. pertussis with 100% specificity. All other laboratories, which used IS481-based assays, reported positive results for the samples containing B. holmesii and B. bronchiseptica, species that have occasionally been recovered from human respiratory samples. These data show that the choice of the target gene is particularly critical for the species specificity of B. pertussis PCR assays.

Bordetella pertussis↗
PubMed2001

Development of an enzyme-linked immunosorbent assay for serotyping ureaplasma urealyticum strains using monoclonal antibodies.

Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains of U. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.

Antibodies, Bacterial↗
PubMed2001

Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula.

We describe an outbreak of necrotizing enterocolitis (NEC) that occurred in the neonatal intensive care unit of our hospital. A total of 12 neonates developed NEC in June-July 1998. For two of them, twin brothers, the NEC turned out to be fatal. Enterobacter sakazakii, a known contaminant of powdered milk formula, was isolated from a stomach aspirate, anal swab, and/or blood sample for 6 of the 12 neonates. A review of feeding procedures revealed that 10 of the 12 patients were fed orally with the same brand of powdered milk formula. E. sakazakii was isolated from the implicated prepared formula milk as well as from several unopened cans of a single batch. Molecular typing by arbitrarily primed PCR (AP-PCR) confirmed, although partially, strain similarity between milk and patient isolates. No further cases of NEC were observed after the use of the contaminated milk formula was stopped. With this outbreak we show that intrinsic microbiological contamination of powdered milk formula can be a possible contributive factor in the development of NEC, a condition encountered almost exclusively in formula-fed premature infants. The use of sterilized liquid milk formula in neonatal care could prevent problems with intrinsic and extrinsic contamination of powdered milk formula.

Bacterial Typing Techniques↗
PubMed2001

Prevalence of genotypic resistance among antiretroviral drug-naive HIV-1-infected patients in Belgium.

OBJECTIVES: To estimate the prevalence and the evolution over time (1995-1998) of genotypic resistance to antiviral drugs in antiretroviral drug-naive HIV-1-infected patients in Belgium. DESIGN: Belgian Aids Reference Laboratories provided retrospective samples and clinical data from antiretroviral drug-naive HIV-1-infected patients who visited the hospital for the first time in 1995 (n=45), 1997 (n=75) and 1998 (n=111). Genotypic resistance to the three available classes of drugs was monitored using the Line Probe Assay (Innogenetics, Gent, Belgium). Additionally, ARMS-151 was performed for scoring multinucleoside resistance. RESULTS: The prevalence of genotypic resistance at baseline to nucleoside analogue reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were each between 10% and 20% for 1995, 1997 and 1998 without an increasing trend over time. For NRTIs, resistance mutations were mainly related to zidovudine in 1995, whereas in 1997 and 1998 baseline resistance was scored for zidovudine, lamivudine or for both drugs simultaneously. No patients displayed the multi-nucleoside resistance Q151M mutation. Baseline resistance mutations to protease inhibitors (PIs) did not rise significantly: 4.4% in 1995, 8% in 1997 and 9.9% in 1998. When scoring any resistance-related mutation, 26.6% displayed genotypic baseline resistance in 1995, 26.6% in 1997 and 31.5% in 1998. DISCUSSION: The prevalence of genotypic baseline resistance to any drug, as scored with LiPA, in naive HIV-1 patients in Belgium is 29%, with baseline resistance mutations to one or several drugs from all available classes of antiviral drugs. The ability of LiPA to pick up minor variants could be an explanation for the higher overall prevalence we observe, when compared to recent estimates in other countries of 16.3% and 22%, which were based on sequencing methods. According to the European guidelines for resistance testing, resistance testing in Belgium before starting antiviral therapy should be considered.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Baseline HIV type 1 genotypic resistance to a newly added nucleoside analog is predictive of virologic failure of the new therapy.

We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.

CD4 Lymphocyte Count↗
PubMed2000

Blinded, multicenter quality control study for the quantification of human immunodeficiency virus type 1 RNA in plasma by the Belgian AIDS reference laboratories.

OBJECTIVE: In order to evaluate the interlaboratory variation of HIV-1 RNA measurements in plasma, the Belgian AIDS reference laboratories organized a blinded multicenter quality control study. METHODS: Atest panel of coded spiked HIV-1 plasma samples reflecting the dynamic range of the assay was composed and distributed. The HIV-1 RNA concentration of these samples was determined by the eight Belgian AIDS reference laboratories by means of the Amplicor HIV-1 Monitor version 1.5 assay. RESULTS: Analysis of the results demonstrated that there was little interlaboratory variation for the high concentration range (4.0-5.7 log10 copies/mL), never exceeding 0.2 log10 copies/mL. However the standard deviation for the low concentration range (2.6-3.9 log10 copies/mL) reached up to 0.22 log10 copies/mL. CONCLUSIONS: Since interlaboratory variability never reached 0.5 log10 copies/mL and each of the laboratories was able to detect four-fold differences in plasma HIV-1 RNA levels, the Amplicor assay can be used in multicenter studies without a centralized analysis of samples. Furthermore, this well-characterized proficiency panel of spiked plasma samples could be used as a standard in the study of interassay comparisons.

Belgium↗
PubMed2000

Development of monoclonal antibodies against Ureaplasma urealyticum serotypes and their use for serotyping clinical isolates.

Monoclonal antibodies (MAbs) against Ureaplasma urealyticum serotype 2, 5, 7, 8, 10, 11, 12, and 13 reference strains were developed. The reactivities of these MAbs with the 14 serotype reference strains was verified by colony immunofluorescence assay and Western blot assay. MAbs against serotypes 2, 7, 10, 11, and 12 were serotype specific, whereas MAbs against serotypes 5, 8, and 13 showed cross-reactivity. All MAbs against serotype 5 were cross-reactive with serotype 2, and one showed, in addition, cross-reactivity to serotypes 9 and 10. Mutual cross-reactivities were observed between MAbs against serotypes 8 and 13. The usefulness of the MAbs for the serotyping of U. urealyticum strains was evaluated by serotyping 21 selected clinical isolates. A complete set of MAbs (the newly developed MAbs and the previously described MAbs against serotypes 1, 3, 4, 6, 9, and 14) as well as a complete set of polyclonal antibodies (PAbs), PAbs 1 to 14, were used. MAbs were able to identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more valuable tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity.

Antibodies, Bacterial↗
PubMed1998

Neonatal infections with Pseudomonas aeruginosa associated with a water-bath used to thaw fresh frozen plasma.

In our 15-bed neonatal intensive care unit (NICU), four new-borns were found to be colonized or infected with Pseudomonas aeruginosa within a period of one week. To identify the outbreak source, three independent studies were performed: epidemiological investigation, environmental surveillance and genotypic typing of isolates. Although epidemiological investigation by a case-control study revealed no conclusive results, the transfusion of fresh frozen plasma (FFP) and human albumin (HA) appeared to be the factor with highest risk. Environmental surveillance and random amplification of polymorphic DNA (RAPD) of isolates identified a water-bath used to warm FFP and HA as the likely reservoir for the outbreak. Further spread of the organism did not occur after elimination of this water-bath from the NICU. RAPD identified in addition an isolate from an infant hospitalized in the NICU five months before the outbreak with a pattern matching the one of the outbreak cluster.

Belgium↗
PubMed1998

Identification of new verocytotoxin type 2 variant B-subunit genes in human and animal Escherichia coli isolates.

The sequence of a verocytotoxin 2 (VT2) variant gene that was untypeable by the B subunit PCR and restriction fragment length polymorphism analysis (PCR-RFLP) method described by Tyler et al. (S. D. Tyler, W. M. Johnson, H. Lior, G. Wang, and K. R. Rozee, J. Clin. Microbiol. 29:1339-1343, 1991) was determined and compared with published sequences. It was highly homologous to two recently reported VT2 variant sequences. The PCR-RFLP method described by Tyler et al. was extended to include these new sequences. New VT2 variants were identified in 65 of 359 VT-producing Escherichia coli (VTEC) with newly designed primers (VT2-cm and VT2-f) and were characterized as well by restriction analysis of the amplification products obtained with another VT2-specific primer pair (VT2-e and VT2-f). The VT genes harbored by 64 of these isolates proved to be untypeable by Tyler's PCR-RFLP method because no amplification was obtained with the primers used with this method (VT2-c and VT2-d). The last isolate harbored the new variant gene in addition to VT2vh-a. None of the isolates harboring these new toxin genes belonged to serogroups O157, O26, O103, O111, and O145. All 65 isolates were negative for the eaeA gene and were significantly less frequently enterohemolytic or positive for the enterohemorrhagic E. coli (EHEC) virulence plasmid than non-O157 VTEC isolates harboring other VT2 genes. They were also less frequently isolated from patients with EHEC-associated symptoms. The extended PCR-RFLP typing method is a useful tool to identify less-virulent VTEC isolates and for VT genotyping in epidemiological studies with non-O157 strains.

Animals↗
PubMed1996

Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene.

HIV-1 and related viruses require co-receptors, in addition to CD4, to infect target cells. The chemokine receptor CCR-5 (ref.1) was recently demonstrated to be a co-receptor for macrophage-tropic (M-tropic) HIV-1 strains, and the orphan receptor LESTR (also called fusin) allows infection by strains adapted for growth in transformed T-cell lines (T-tropic strains). Here we show that a mutant allele of CCR-5 is present at a high frequency in caucasian populations (allele frequency, 0.092), but is absent in black populations from Western and Central Africa and Japanese populations. A 32-base-pair deletion within the coding region results in a frame shift, and generates a non-functional receptor that does not support membrane fusion or infection by macrophage- and dual-tropic HIV-1 strains. In a cohort of HIV-1 infected caucasian subjects, no individual homozygous for the mutation was found, and the frequency of heterozygotes was 35% lower than in the general population. White blood cells from an individual homozygous for the null allele were found to be highly resistant to infection by M-tropic HIV-1 viruses, confirming that CCR-5 is the major co-receptor for primary HIV-1 strains. The lower frequency of heterozygotes in seropositive patients may indicate partial resistance.

Alleles↗
PubMed1996

Expression in E. coli and purification of the active autoprotease P20 of classical swine fever virus.

The genes for Flaviviridae structural proteins are located at the 5' terminus of the genome, while the 3' terminus contains the genes for the non-structural proteins. The first protein product of the large ORF of pestiviruses, the p20 protein, is however a non-structural protein which possess an autoproteolytic activity. Here we report the cloning of the p20/p14 genes behind the strong Trc promotor and expression of the p20 at high levels in E. coli. The autoprotease p20 was responsible for its own release from the nascent polyprotein in E. coli and was further purified by chromatography techniques.

Animals↗
PubMed1993

Serological and antigenical findings indicating pestivirus in man.

An epidemiological survey for pestivirus was undertaken in Zambia and Europe, in view of the recent serological findings obtained by previous studies in Europe with humans. Collected sera were tested for anti-bovine viral diarrhea virus (BVDV) specific antibodies by IIF and Western Blotting. Of those individuals tested (n = 1272), 15.3% showed a seropositive reaction to the BVDV. Anti-BVDV antibody prevalence in immuno-depressed patients (e.g. HIV positive) was investigated. A higher prevalence was revealed in HIV patients suffering from chronic diarrhoea and in those having developed AIDS Related Complex (ARC). Our of 212 persons tested for pestivirus isolation, a non cytopathic virus strain was detected in 2 buffy coat samples using IIF with a specific anti-BVDV serum. The isolation could be repeated three times during 31 days in one person. The virus was identified as a pestivirus with radioimmuno-precipitation assays and IIF-flow cytometry. A doublet of 120 kD was identified only in cell lysates, indicating a non-structural protein. In order to rule out cross reactivity 30 sera from Hepatitis C seropositive patients were tested against the isolate by IIF-flow cytometry. No antigen-specific binding could be observed. These findings indicated the occurrence of a pestivirus in man and might suggest a relationship with a pestivirus of animal origin.

Animals↗
PubMed1993

Polymerase chain reaction-mediated cloning and in vitro translation of the genes coding for the structural proteins of hog cholera virus.

After amplification by PCR, the 5' region of the genome of hog cholera virus (HCV) strain Alfort 187 was cloned and sequenced. The nucleotide and deduced amino acid sequences were compared with the ones of other pestiviruses. By in vitro translation experiments we were able to demonstrate the protease activity of the p 20 protein of HCV.

Base Sequence↗
PubMed1993

Characterization of structural and non-structural proteins of hog cholera virus by means of monoclonal antibodies.

A panel of 15 monoclonal antibodies, produced against the hog cholera virus, were characterized by radioimmunoprecipitation assays. Using this panel, we were able to identify 4 sets of monoclonal antibodies precipitating each a different viral protein with relative molecular weight of 40, 46, 120 kDa, respectively, and a protein complex containing 15, 16, 27, and 55 kDa polypeptides which were further characterized. One monoclonal antibody recognized an antigenic determinant at the C-terminal cleavage product of the non-structural p 125 of BVDV. The 40 kDa protein was precipitated from the pelleted virions, indicating its structural importance. On the contrary the 46 kDa protein could only be precipitated from the cell lysate and not from the pelleted virions. The glycosylated 15/16 kDa-55 kDa proteins form a disulfide linked heterodimer on the virus particle with a relative molecular weight of 65 kDa.

Animals↗