Browsing by Author "Hoek, Kim Gilberte Pauline"
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- ItemInvestigation into genotypic diagnostics for mycobacterium tuberculosis(Stellenbosch : Stellenbosch University, 2010-12) Hoek, Kim Gilberte Pauline; Warren, Robin Mark; Van Helden, Paul David; Walzl, Gerhard; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Diagnostic delay is regarded as a major contributor to the continuous rise in tuberculosis (TB) cases and the emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). It is therefore essential that more rapid diagnostic methods are developed. Molecular-based assays have the potential for the rapid species-specific diagnosis of TB and associated drug-resistances directly from clinical specimens. We investigated whether high resolution melting analysis (HRM) could enable the rapid diagnosis of TB and associated drug resistance, since the HRM apparatus and reagents are relatively inexpensive and the methodology can easily be implemented in high incidence, low income regions. Application of this methodology allowed for the rapid identification of mycobacterial lymphadenitis from fine-needle aspiration biopsy (FNAB) samples in 2 studies. This was done by targeting the region of deletion 9 (RD9), present in M. tuberculosis and M. canettii, but absent from all other members of the complex. However, the sensitivity of the method was low (51.9% and 46.3%, respectively) when compared to the reference standard (positive cytology and/or positive culture). Despite this limitation our method was able to provide a rapid diagnosis in more than half of the infected patients with a relatively high specificity (94.0% and 83.3%, respectively). We therefore proposed a diagnostic algorithm allowing the early treatment of patients with both HRM and cytology results indicative of mycobacterial disease. We developed the Fluorometric Assay for Susceptibility Testing of Rifampicin (FAST-Rif) which allowed the rapid diagnosis of MDR-TB by detecting rifampicin (RIF) resistance mutations in the rpoB gene with a sensitivity and specificity of 98% and 100%, respectively. The FAST-Rif method was easily adapted to detect ethambutol (EMB) resistance due to mutations in the embB gene with a sensitivity and specificity of 94.4% and 98.4% respectively, as compared to DNA sequencing. The FAST-EMB method was a significant improvement over the inaccurate culture based method. We identified a strong association between EMB resistance (and pyrazinamide resistance) and MDR-TB and subsequently advised modifications to the current (2008) South African National TB Control Programme draft policy guidelines. Due to the potential for amplicon release, we adapted the FAST-Rif and FAST-EMB methods to a closed-tube one-step method using the detection of inhA promoter mutations conferring isoniazid (INH) resistance as a model. The method (FASTest-inhA) was able to identify inhA promoter mutations with a sensitivity and specificity of 100% and 83.3%. These mutations are of particular interest as they confer low level INH resistance and cross-resistance to ethionamide (Eto). Since inhA promoter mutations are strongly associated with XDR-TB in the Western and Eastern Cape Provinces of South Africa, data generated by the recently implemented GenoType® MDRTBPlus assay may allow individualised treatment regimens to be designed for a patient depending on their INH mutation profile. Our proposed treatment algorithm may be particularly useful in XDR-TB cases, for which only few active drugs remain available. Since current diagnostic methods all carry advantages and disadvantages, a combination of phenotypic and genotypic-based methodologies may be the best scenario while awaiting superior methods.