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1.  Confirmation diagnosis of influenza A(H1N1)2009 by Belgian sentinel laboratories during the epidemic phase 
Archives of Public Health  2010;68(2):76-82.
PMCID: PMC3463024
Influenza; A(H1N1)pandemic; epidemiology; laboratory diagnosis
3.  The Impact of Individual Human Immunodeficiency Virus Type 1 Protease Mutations on Drug Susceptibility Is Highly Influenced by Complex Interactions with the Background Protease Sequence▿ †  
Journal of Virology  2009;83(18):9512-9520.
The requirement for multiple mutations for protease inhibitor (PI) resistance necessitates a better understanding of the molecular basis of resistance development. The novel bioinformatics resistance determination approach presented here elaborates on genetic profiles observed in clinical human immunodeficiency virus type 1 (HIV-1) isolates. Synthetic protease sequences were cloned in a wild-type HIV-1 background to generate a large number of close variants, covering 69 mutation clusters between multi-PI-resistant viruses and their corresponding genetically closely related, but PI-susceptible, counterparts. The vast number of mutants generated facilitates a profound and broad analysis of the influence of the background on the effect of individual PI resistance-associated mutations (PI-RAMs) on PI susceptibility. Within a set of viruses, all PI-RAMs that differed between susceptible and resistant viruses were varied while maintaining the background sequence from the resistant virus. The PI darunavir was used to evaluate PI susceptibility. Single sets allowed delineation of the impact of individual mutations on PI susceptibility, as well as the influence of PI-RAMs on one another. Comparing across sets, it could be inferred how the background influenced the interaction between two mutations, in some cases even changing antagonistic relationships into synergistic ones or vice versa. The approach elaborates on patient data and demonstrates how the specific mutational background greatly influences the impact of individual mutations on PI susceptibility in clinical patterns.
PMCID: PMC2738252  PMID: 19587054
4.  Production of Recombinant Antigens of Ureaplasma parvum Serotypes 3 and 6 for Development of a Serological Assay▿  
Recombinant antigens of Ureaplasma parvum serotypes 3 and 6 were produced in order to develop a serological assay for Ureaplasma antibody detection. The genes of the multiple banded antigen (MBA) were amplified by PCR and cloned in a pTrcHis TOPO plasmid. Purified recombinant proteins were evaluated in Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies and human sera. Our approach was successful in the production of the recombinant MBAs (rMBAs) for serotypes 3 and 6. The antigens tested positive with serotype-specific monoclonal antibodies in Western blotting and in ELISA. Prominent reactions were detected with the rMBAs and their homologous monoclonal antibodies. Strong cross-reactions were visible in ELISA between rMBA 3 and the monoclonal antibodies from the other U. parvum serotypes. A weak cross-reaction was seen with rMBA 3 and the monoclonal antibody from serotype 4. rMBA 6 showed cross-reaction only with the monoclonal antibody from U. parvum serotype 1. Fifty-one percent of the sera obtained from culture-positive women reacted with one or both rMBAs. Only three (15%) of the sera from culture-negative women reacted with the rMBA. The positive reactions were observed only with rMBA 6. These preliminary tests showed the potential usefulness of the rMBAs produced for detecting an antibody response against Ureaplasma antigens.
PMCID: PMC2268256  PMID: 18094114
5.  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.
PMCID: PMC540137  PMID: 15634947
7.  Identification of New Verocytotoxin Type 2 Variant B-Subunit Genes in Human and Animal Escherichia coli Isolates 
Journal of Clinical Microbiology  1998;36(11):3317-3322.
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.
PMCID: PMC105321  PMID: 9774585

Results 1-7 (7)