Biomedical subjects

Paul van Helden

Publications and source records attributed to Paul van Helden.

10 recordsLinked to original sources

PubMed2007

Absence of an association between Mycobacterium tuberculosis genotype and clinical features in children with tuberculous meningitis.

OBJECTIVES: Animal studies point to increased virulence of certain mycobacterial strains, notably those of the Beijing genotype. There are limited data on mycobacterial genotypic diversity in children with tuberculous meningitis (TBM). We investigated mycobacterial genotypic diversity in children with TBM and analyzed the relationship among genotype, clinical presentation and outcome. PATIENTS AND METHODS: Data were extracted from an ongoing prospective study on children with confirmed TBM from 1992 through 2003 at a referral hospital in the Western Cape Province, South Africa. Mycobacterial isolates were genotyped by standardized restriction fragment length polymorphism methodology. Clinical data at diagnosis, inflammatory progression during the first month of antituberculosis therapy and neurologic outcomes after 6 months of therapy were analyzed according to the principal genetic group of the strain and the presence of the Beijing strain, respectively. RESULTS: Fifty-nine children were included (median age at diagnosis, 23 months); 37 presented with stage II and 22 with stage III presented with TBM. At completion of antituberculosis therapy, 6 children were neurologically normal, 22 were moderately neurologically impaired, 23 were severely neurologically impaired and 6 children died; detailed outcomes were not available in 2 children. All 3 principal genetic groups were represented (group 1, 27.1%; group 2, 59.3%; group 3, 13.6%); the most prevalent strains were of the Beijing genotype (family 29; 25.4%), followed by family 28 (10.2%) and family 11 (8.5%). Predictors of poor neurologic outcome included advanced disease at diagnosis and male gender. There was no association between the principal genetic group of the strain or the presence of the Beijing genotype, and clinical presentation or outcome. CONCLUSIONS: We found no association between Mycobacterium tuberculosis genotypes and clinical presentation or outcome.

Adolescent↗

PubMed2006

Safe Mycobacterium tuberculosis DNA extraction method that does not compromise integrity.

Concern has been raised about the efficacy of the heat killing of mycobacteria during the isolation of DNA. We demonstrate a method that allows for the efficient killing of Mycobacterium tuberculosis without compromising DNA integrity for subsequent molecular investigation. This method reduces the risk of infection to the research scientist.

Bacteriological Techniques↗

PubMed2005

A rapid method of diagnosing pulmonary tuberculosis using stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry.

A fast method for detection of tuberculostearic acid (TBSA) in sputum samples is described. The samples, obtained from patients with known or suspected pulmonary tuberculosis, were decontaminated and concentrated before being analyzed by stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry (SBSE-TD-GC-MS). Prior to extraction, the mycobacterial lipids were hydrolyzed and then derivatized with ethyl chloroformate to increase the sorption of the compounds by the polydimethylsiloxane (PDMS) stir bar coating. The limit of detection (LOD) is 0.2 ng ml(-1). Four sputum samples that were classified by direct microscopy as smear-positive or negative were analyzed by GC-MS. TBSA was detected at concentrations ranging from 0.47 to 2.3 ng ml(-1). The method is sufficiently sensitive to detect TBSA directly in clinical samples without the need to culture the organisms.

Gas Chromatography-Mass Spectrometry↗

PubMed2005

TB: a partnership for the benefit of research and community.

A public-private partnership (PPP) involving Stellenbosch University in South Africa and GlaxoSmithKline (GSK) has benefited both research and a local community where tuberculosis (TB) is endemic. The venture, part of GSK's Action TB programme, enabled the University's Desmond Tutu TB Centre to establish an epidemiological field site in two suburbs of Cape Town where the annual risk of TB infection is 3.5%. Collaboration between the centre and GSK focused on the development of a surrogate marker model able to predict patient outcome with relative accuracy. Such models may be useful tools for diagnosis/prognosis and for shortening clinical trials of novel TB agents. Other research findings stemming from the Action TB partnership suggest that exogenous reinfection is responsible for the majority of relapse cases and that adults often have infection with multiple strains. The local community has been empowered by the implementation of the Directly Observed Treatment, Short-course (DOTS) programme and benefited from improved education about health in general and TB in particular. The centre has also provided employment for many local people in field work and other roles. Meanwhile, national and international publicity about the centre's work has aided in generating the essential political will to allocate resources and shape healthcare priorities, benefiting this impoverished community.

Adult↗

PubMed2005

Protein expression by a Beijing strain differs from that of another clinical isolate and Mycobacterium tuberculosis H37Rv.

The Beijing strain family has often been associated with tuberculosis (TB) outbreaks and drug resistance worldwide. In this study the authors have compared the protein expression and antigen recognition profiles of a local Beijing strain with a less prevalent clinical isolate belonging to the family 23 strain lineage, and the laboratory strain Mycobacterium tuberculosis H37Rv. Using two-dimensional electrophoresis, liquid chromatography tandem mass spectrometry and Western blot analysis several proteins were identified as quantitatively increased or decreased in both clinical strains compared to H37Rv. Remarkably, the Beijing strain showed increased expression of alpha-crystallin and decreased expression of Hsp65, PstS1, and the 47 kDa protein compared to the other clinical strain and H37Rv. One- and two-dimensional Western blot analysis of antigens expressed by the three strains, using plasma from TB patients, confirmed differential antigen expression by strains and patient-to-patient variation in humoral immunity. These observed protein differences could aid the elucidation of mechanisms underlying the success of the Beijing strain family, measured by global dissemination, compared to other M. tuberculosis strains.

Animals↗

PubMed2004

Differential expression of mycothiol pathway genes: are they affected by antituberculosis drugs?

Mycothiol (MSH) is the major cellular thiol in Mycobacterium tuberculosis (M.tb). We hypothesize that the mycothiol-dependent detoxification pathway may serve an important role during oxygen stress management in M. tuberculosis, derived from normal aerobic metabolism, the macrophage environment and through the action of anti-tubercular antibiotics, such as Isoniazid (INH). Total mRNA and DNA were isolated from M. bovis BCG at different stages of growth in 7H9 mycobacterial medium. Three genes involved in mycothiol metabolism and encoding the enzymes mycothiol S-conjugate amidase (Mca, Rv1082), NADPH dependent mycothiol reductase (mtr, Rv2855), and N-Acetyl-1-D-myo-Inosityl-2-Amino-2-Deoxy-alpha-D-Glucopyranoside Deacetylase (GlcNAc-Ins deacetylase, Rv1170 or mshB) were investigated for genomic rearrangements and expression. The results show that the genomic domains of the genes remain conserved in evolutionary diverse and unrelated M. tuberculosis isolates. The genes encoding enzymes implicated in mycothiol reduction, mtr (Rv2855) and the mycothiol-dependant detoxification of electrophilic agents, Mca (Rv1082), are shown to be actively transcribed during logarithmic M. bovis BCG growth. The gene encoding GlcNAc-Ins deacetylase (the rate limiting mycothiol biosynthesis step) shows induction in the presence of INH. Antisense oligonucleotides to both GlcNAc-Ins deacetylase (Rv1170) and mtr (Rv2855) mRNA affect mycobacterial growth. In conclusion the results presented here suggest that these enzymes are sensitive to free radical generating antituberculosis drugs and may be useful targets for new drug development.

Acetylglucosamine↗

PubMed2004

Resistant Mycobacterium bovis Bacillus Calmette-Guérin disease: implications for management of Bacillus Calmette-Guérin Disease in human immunodeficiency virus-infected children.

Guidelines for the diagnosis and management of Bacillus Calmette-Guérin (BCG) disease in children are lacking, and there are limited data on drug resistance of Mycobacterium bovis BCG. A 6-month-old HIV-infected infant presented with right axillary adenitis ipsilateral to the site of BCG immunization. M. tuberculosis complex was cultured from axillary lymph nodes and gastric aspirates, and M. bovis BCG was isolated. Susceptibility testing before initiation of therapy demonstrated inherent resistance to isoniazid. The organism acquired rifampin resistance during therapy. This was confirmed by the presence of a mutation in codon 531 (Ser531Tyr) of the rpoB gene. Treatment guidelines for BCG disease with consideration of inherent and possible acquired drug resistance should be established in settings with high rates of vertical HIV transmission and routine BCG vaccination.

Animals↗

PubMed2003

Immunophenotyping of macrophages in human pulmonary tuberculosis and sarcoidosis.

Classic studies of tuberculosis (TB) revealed morphologic evidence of considerable heterogeneity of macrophages (MØs), but the functional significance of this heterogeneity remains unknown. We have used newly available specific antibodies for selected membrane and secretory molecules to examine the phenotype of MØs in situ in a range of South African patients with TB, compared with sarcoidosis. Patients were human immunodeficiency virus-negative adults and children, and the examined biopsy specimens included lung and lymph nodes. Mature pulmonary MØs (alveolar, interstitial, epithelioid and multinucleated giant cells) selectively expressed scavenger receptor type A and a novel carboxypeptidase-like antigen called carboxypeptidase-related vitellogenin-like MØ molecule (CPVL). CPVL did not display enhanced expression in sarcoidosis, vs. TB patients, as observed with angiotensin-converting enzyme (ACE), a related molecule. Immunocytochemical studies with surfactant proteins (SP)-A and -D showed that type II alveolar cells expressed these collectins, as did MØs, possibly after binding of secreted proteins. Studies with an antibody specific for the C-terminus of fractalkine, a tethered CX3C chemokine, confirmed synthesis of this molecule by bronchiolar epithelial cells and occasional endothelial cells. These studies provide new marker antigens and extend previous studies on MØ differentiation, activation and local interactions in chronic human granulomatous inflammation in the lung.

Adult↗

PubMed2002

Fine mapping of a putative tuberculosis-susceptibility locus on chromosome 15q11-13 in African families.

Host genetics plays an important role in individual susceptibility and resistance to infectious diseases, but no genes have yet been identified using genome-wide screens. Twin studies have indicated that tuberculosis susceptibility has a significant host genetic component, and several genes appear to be involved. Recently, a genome-wide linkage analysis of 136 African families identified chromosome 15q11-13 as a region with suggestive evidence of linkage, with a LOD score of 2.0. We tested 10 microsatellite markers and 5 positional candidate genes in this chromosomal region for deviation from random transmission from parents to affected offspring. The polymorphisms, lying in a region of 14 cM, were initially typed in the same 79 Gambian families used in the genome screen. A borderline significant association with a 7 bp deletion in UBE3A (P = 0.01) was found. This polymorphism was then evaluated further in a larger series of families with tuberculosis, including 44 Guinean families and 57 families from South Africa. Testing for association between the deletion and tuberculosis across all the families using the exact symmetry test further supported the association (overall P = 0.002). These fine-mapping data suggest that UBE3A or a closely flanking gene may be a tuberculosis-susceptibility locus.

Black People↗

PubMed2002

Protein expression in Mycobacterium tuberculosis differs with growth stage and strain type.

Different phenotypes are displayed by Mycobacterium tuberculosis (M. tuberculosis) strains, fuelling speculation that certain strains are "hypervirulent" and able to evade host defenses better than others. Furthermore, differential antigen expression by M. tuberculosis strains may explain why certain patients are susceptible to a repeat episode of tuberculosis. The objective of this study was to compare protein expression by M. tuberculosis H37Rv and clinical isolates in order to determine whether differential protein expression contributes to the different phenotypes expressed by these strains. Expression of alpha-crystallin, the antigen 85 complex, PstS-1, L-alanine dehydrogenase and the 65 kDa antigen was analysed by Western blotting and enzyme-linked immunosorbent assays, using mouse monoclonal antibodies. We found no significant difference in the growth rate of the M. tuberculosis strains in vitro, and although M. tuberculosis protein expression showed phase variation during growth, expression seemed to be qualitatively, but not quantitatively, conserved in the strains investigated. These results have potentially important implications for vaccine development and serodiagnosis.

Animals↗