Conventional drug susceptibility testing techniques, the ‘gold standard’ for M.
tuberculosis are slow, requiring about 3-6 weeks from a positive culture. This
diagnostic delay, before initiation of appropriate treatment, contributes to increased
transmission rates. Molecular techniques provide rapid results and therefore present
an alternative to conventional tests. The aim of this project was to develop an inhouse
reverse line blot hybridization assay (RIFO assay) that could detect mutations
associated with Rifampicin resistance directly in clinical specimens of patients in
KwaZulu Natal.
A 437 bp region of the rpoB gene was sequenced to ascertain the most frequently
occurring mutations conferring resistance to rifampicin in isolates in KwaZulu-Natal.
Wildtype and mutant probes designed to target these mutations, were immobilized on
a Biodyne C membrane. Hybridization conditions were optimized using biotin labeled
PCR products from culture. Detection was performed with peroxidase labeled
streptavidin using enhanced chemiluminescence. Four DNA extraction methods were
evaluated on sputum specimens to determine the one with the least inhibitory effect
on amplification.
A total of 11 mutations were found in 236 clinical isolates: 531TTG (109, 58.3%),
516GTC (26, 13%), 533CCG/516GGC (20, 10%), 533CCG (18, 9.6%), other
mutations < 5% each. The chelex extraction method was found to be optimal for
removing inhibitors in sputum specimens. Sputum specimens of 404 patients
hospitalized at King George V Hospital between 2005 and 2006 were rifoligotyped.
The RIFO assay was optimised on clinical isolates and then applied to sputum
specimens. The RIFO assay on culture and sputum correlated well with the DST
(sensitivity 92% and 94% respectively). However, the specificity was very low in
both culture and sputum specimens compared to DST (38% and 35% respectively).
This could be attributed to the presence of silent mutations, mixed infections, mixed
populations of bacteria or the small number of susceptible strains used in this study.
The in-house RIFO assay can be used directly on sputum specimens to predict
Rifampicin resistance and therefore MDR-TB in less than a week compared to the
gold standards. A total of 43 samples can be tested simultaneously at low cost and the
membrane is reusable compared to commercial kits such as the Hains test that is
expensive and strips are not reusable. A similar assay can be designed to target
mutations for the detection of XDR-TB. Future studies should be conducted in a
clinical setting on patients with sensitive strains to increase the specificity. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/5850 |
| Date | January 2011 |
| Creators | Ndimande, Bongiwe Olga. |
| Contributors | Pillay, M. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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