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K A De Smet

Publications and source records attributed to K A De Smet.

14 recordsLinked to original sources
PubMed2001

Laboratory guidelines for the practical use of HIV drug resistance tests in patient follow-up.

HIV drug resistance is one of the major limitations in the successful treatment of HIV-infected patients using currently available antiretroviral combination therapies. When appropriate, drug susceptibility profiles should be taken into consideration in the choice of a specific combination therapy. Guidelines recommending resistance testing in certain circumstances have been issued. Many clinicians have access to resistance testing and will increasingly use these results in their treatment decisions. In this document, we comment on the different methods available, and the relevant issues relating to the clinical application of these tests. Specifically, the following recommendations can be made: (i) genotypic and phenotypic HIV-1 drug resistance analyses can yield complementary information for the clinician. However, insufficient information currently exists as to which approach is preferable in any particular clinical setting; (ii) when HIV-1 drug resistance testing is required, it is recommended that testing be performed on plasma samples obtained before starting, stopping or changing therapy, on samples that have a viral load above the detection limit of the resistance test; (iii) the panel recommends that genotypic and phenotypic HIV-1 drug resistance testing for clinical purposes be performed in a certified laboratory under strict quality control and quality assurance standards; and (iv) the panel recommends that resistance testing laboratories provide clinicians with resistance reports that include a list of drug-related resistance mutations (genotype) and/or a list of drug-related fold resistance values (phenotype), with interpretations of each by an experienced virologist. The interpretation of genotypic and phenotypic analysis is a complex and developing science, and in order to understand HIV-1 drug resistance reports, communication between the requesting clinician and the expert that interpreted the resistance report is recommended.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Relationship between pyrazinamide resistance, loss of pyrazinamidase activity, and mutations in the pncA locus in multidrug-resistant clinical isolates of Mycobacterium tuberculosis.

Sixty-two Mycobacterium tuberculosis isolates were tested for pyrazinamidase activity, and their pyrazinamide susceptibility was determined by the radiometric method. Sequencing of pncA genes in the 23 resistant strains revealed mutations in 16 pyrazinamidase-negative strains, 11 of which had not been previously described. Six isolates containing wild-type pncA might possess alternative resistance mechanisms.

Amidohydrolases↗
PubMed1998

Human T- and B-cell reactivity to the 16kDa alpha-crystallin protein of Mycobacterium tuberculosis.

The attributes of immunodominance, predominant expression during mycobacterial dormancy and restriction to the Mycobacterium tuberculosis complex make the 16 kDa protein an important candidate for the study of the immune response in humans. We therefore investigated the relationship between T- and B-cell reactivity to the recombinant antigen and disease in a total of 127 subjects. The percentage of T-cell responders towards both the intact antigen and its permissively recognised peptide 16p91-110 was highest in healthy bacillus Calmette-Guérin (BCG)-sensitized controls (96% and 68%, respectively) and lowest in those with extensive untreated tuberculosis (26% and 18%) (P < 0.001). By contrast, antibody levels (ABT50 > 100) were highest in patients with extensive disease (46-50%) (P < 0.005). There was significantly higher production of IFN-gamma in the BCG-sensitized controls by comparison with untreated patients (P < 0.05), but complete antituberculous chemotherapy abolished this deficit in patients. The significance of these findings to immunodiagnosis and protective immunity is discussed.

Antigen Presentation↗
PubMed1998

Variation amongst human isolates of Brachyspira (Serpulina) pilosicoli based on biochemical characterization and 16S rRNA gene sequencing.

Brachyspira pilosicoli (formerly Serpulina pilosicoli) causes swine spirochaetosis and can also be isolated fro human faeces, although its role in human disease remains unclear. The genetic and biochemical variations amongst 19 isolates of human spirochaetes from five different countries were evaluated and compared to those found amongst swine isolates of B. pilosicoli. All isolates were negative for beta-glucosidase and all but one were positive for hippurate hydrolysis, which are characteristics typical of B. pilosicoli. The isolates showed variation in indole production and alpha-galactosidase and alpha-glucosidase activity, other characteristics which can be used to identify B. pilosicoli. The DNA sequences of part of the 16S rRNA gene differed from each other and from that of B. pilosicoli by 0-3 bp out of 283 bp. It is concluded that there is considerable variation amongst human intestinal spirochaetes. Since few of the isolates reported here match the current criteria for B. pilosicoli, it is concluded that this species is more heterogeneous than previously appreciated. However, it cannot be excluded that some isolates may belong to uncharacterized related Brachyspira/Serpulina species.

Animals↗
PubMed1998

Molecular cloning and functional analysis of a novel tetracycline resistance determinant, tet(V), from Mycobacterium smegmatis.

The nucleotide sequence and mechanism of action of a tetracycline resistance gene from Mycobacterium smegmatis were determined. Analysis of a 2.2-kb sequence fragment showed the presence of one open reading frame, designated tet(V), encoding a 419-amino-acid protein (molecular weight, 44,610) with at least 10 transmembrane domains. A database search showed that the gene is homologous to membrane-associated antibiotic efflux pump proteins but not to any known tetracycline efflux pumps. The steady-state accumulation level of tetracycline by M. smegmatis harboring a plasmid carrying the tet(V) gene was about fourfold lower than that of the parental strain. Furthermore, the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone blocked tetracycline efflux in deenergized cells. These results suggest that the tet(V) gene codes for a drug antiporter which uses the proton motive force for the active efflux of tetracycline. By primer-specific amplification the gene appears to be restricted to M. smegmatis and M. fortuitum.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis.

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.

Amino Acid Sequence↗
PubMed1996

Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare.

Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M intracellulare that is absent in M. avium. This antigen cross reacts immunologically with a major 38 kDa antigen of M. tuberculosis, and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli. Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.

Amino Acid Sequence↗
PubMed1996

Genetic and serovar typing of clinical isolates of the Mycobacterium avium-intracellulare complex.

SETTING: One hundred and thirty-four Mycobacterium avium-intracellulare complex (MAC) isolates were obtained from 121 patients in the UK. OBJECTIVE: To compare serotyping and genetic analysis for species identification of MAC isolates from patients with and without the acquired immunodeficiency syndrome (AIDS). DESIGN: Clinical MAC isolates were cultured and analyzed by serotyping, the commercially available Accuprobe kit, hybridization with genes coding for the 19 kDa and 38 kDa antigens of M. tuberculosis and fingerprinting with the pMB22 probe derived from M. paratuberculosis. RESULTS: Species classification on the basis of genetic analysis was similar to serovar typing, with only exceptional discrepancies. Serovar prevalence was different in the two groups of patients, and different from those reported in other countries. MAC isolates from AIDS patients were exclusively M. avium, whereas patients without AIDS had MAC infections with M. avium and M. intracellulare in about equal proportion. M. intracellulare clinical isolates were genetically more heterogeneous than M. avium. Only M. intracellulare hybridized with the 38 kDa gene probe. CONCLUSIONS: Serovars are strongly linked with species in clinical MAC isolates, confirming results previously obtained with reference strains. M. intracellulare can be easily identified by the presence of a 38 kDa gene.

AIDS-Related Opportunistic Infections↗
PubMed1995

Ribosomal internal transcribed spacer sequences are identical among Mycobacterium avium-intracellulare complex isolates from AIDS patients, but vary among isolates from elderly pulmonary disease patients.

Sequencing 280 bp of the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes in a collection of 46 clinical isolates of the Mycobacterium avium-intracellulare complex (MAI complex) identified nine different sequences, grouping these isolates in nine 'ITS sequevars'. This analysis extends the subdivision within the MAI complex to 18 ITS sequevars and also improves discrimination from other mycobacterial species. Evaluation of the sequevar grouping among different clinical sources revealed strong association of the M. avium sequevar Mav-B with AIDS and with lymphadenitis in children (18 out of 20 and 3 out of 3 respectively). Isolates from elderly patients with pulmonary disease and not suspected of being HIV infected belonged predominantly to M. intracellulare ITS sequevars and sequevars not assigned to either M. avium or M. intracellulare. On the other hand, animal isolates were of both the Mav-A and Mav-B sequevars. We conclude that ITS sequevar typing is an accurate way of identifying distinct MAI complex strains. The observed differences between clinical sources suggest that ITS sequevars reflect possibly important, biologically and clinically relevant polymorphisms between MAI complex organisms.

Acquired Immunodeficiency Syndrome↗
PubMed1994

A versatile system for the production of recombinant chimeric peptides.

Production of chimeric and multimeric peptides is of interest for the analysis of topographic relationships between T and B cell stimulatory epitopes. Recombinant DNA technology has certain advantages over conventional chemical peptide synthesis for the production of peptide constructs of large size (more than 40 amino acid residues). We describe a methodology which is versatile and independent of the expression vector used because it only relies on the incorporation of appropriate restriction enzyme sites in oligonucleotides. The method was verified using two 20mer sequences from the 38 kDa antigen of M. tuberculosis. Peptide 201-220, containing an antibody binding linear epitope, has been made immunogenic in vivo when combined with T cell stimulatory peptide 350-369 in a chimeric peptide. The results demonstrate that a distinct orientation of the constituent peptides was essential for achieving optimal immunogenicity.

Amino Acid Sequence↗
PubMed1994

Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M. tuberculosis.

The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins.

Acetyl-CoA Carboxylase↗
PubMed1993

Tropist3: a cosmid vector for simplified mapping of both G+C-rich and A+T-rich genomic DNA.

We have constructed a cosmid vector, Tropist3, based on the lambda origin double-cos-site vector Lawrist4, which is designed for efficient cloning and mapping of genomic DNA. Tropist3 contains two cloning sites in addition to the HindIII and BamHI sites present in Lawrist4; a SalI site allows cloning of Sau3AI partial digests following partial filling-in of the ends, and a PmlI site is suitable for blunt-end cloning. Both these strategies reduce the chance of co-cloning two inserts. Tropist3 also contains NotI, PacI, SacII and KpnI sites flanking the cloning region; these allow most inserts to be excised cleanly and mapped by partial digestion followed by hybridization with short vector sequences which lie adjacent to the cloning sites. This will also be useful for recloning inserts into different vectors, or for cosmid sequencing projects.

Base Sequence↗
PubMed1993

Sequence and immunological characterization of a serine-rich antigen from Mycobacterium leprae.

Sera from lepromatous leprosy patients were used to screen a Mycobacterium leprae lambda gt11 library. Three positive plaques were picked, and lysogens were constructed. Immunoblot analysis showed that all of the lysogens expressed an apparently identical beta-galactosidase fusion protein which reacted strongly with the sera. The 1.7-kbp insert from one clone was subcloned into the lacZ gene in pUR290; sequence analysis of the end fused to lacZ revealed an open reading frame with no significant homology to previously published sequences. The insert was used to screen an M. leprae cosmid library, and five clones were isolated. The insert was also found to hybridize to clones expressing the M. leprae antigen which had previously been designated class III and 25L. A 1.8-kbp HindIII fragment was subcloned from one of the cosmids and sequenced. The sequence revealed a 1,227-bp open reading frame, encoding a 408-amino-acid protein with a predicted molecular mass of 42,466 Da. The protein contains amino- and carboxy-terminal hydrophobic domains and a hydrophilic central domain; the amino-terminal domain shows some homology to a 51-kDa hypothetical antigen of Mycobacterium tuberculosis, while the hydrophilic region contains a high proportion of serine residues, and we have therefore designated the protein serine-rich antigen (Sra). Some repeated motifs are present in the protein, but their significance is unknown. Seventy-eight percent of serum samples from multibacillary leprosy patients and 68% of serum samples from paucibacillary leprosy patients recognized the fusion protein, showing that this is a major M. leprae antigen. In contrast, all serum samples from endemic controls were negative, while 26% of serum samples from tuberculosis patients were weakly positive.

Amino Acid Sequence↗