Thesis (PhD )--University of Stellenbosch, 2010. / 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. / AFRIKAANSE OPSOMMING: Die onvermoë om tuberkulose (TB), multi-weerstandige tuberkulose (MDR-TB) en uiters
weerstandige tuberkulose (XDR-TB) vinnig te diagnoseer, is ‘n belangrike oorsaak vir die
volgehoue toename en verspreiding daarvan. Dit is noodsaaklik dat diagnostiese toetse wat
vinniger resultate oplewer, ontwikkel word. Molukulêre toetsing het die potensiaal om vinnig
spesie-spesifieke diagnoses van TB en die weerstandigheid teen TB-medikasie te lewer. Hierdie
studie wil vasstel of hoë-resolusie smeltingsanalise (HRS) ‘n vinnige diagnose van TB en die
weerstandigheid teen TB-medikasie kan oplewer aangesien die relatiewe lae koste van reagense
en apparaat, asook die minimale infrastruktuur en vaardighede wat vir dié toets benodig word, dit
uiters geskik maak vir pasiënte in gebiede met ‘n hoë TB-insidensie en lae inkomste.
Die toepassing van die HRS-metode op fynnaald-aspiraatbiopsies in twee afsonderlike studies,
het gelei tot die vinnige identifisering van mikrobakteriële-limfadenitis. Dit is bemiddel deur die
gebied van delesie 9 (RD9) teenwoordig in Mycobacterium tuberculosis en M. canettii, maar
afwesig in al die ander lede van die kompleks, te teiken. Die sensitiwiteit van die metode was
(51.9% en 46.3%, vir die twee studies onderskeidelik) in vergelyking met die verwysingstandaard
(positiewe sitologie en/of positiewe kultuur). Ten spyte van dié beperking was ‘n vinnige
diagnose in meer as die helfte van geïnfekteerde pasiënte met ‘n redelike hoë spesifisiteit
(94.0% en 83.3%, onderskeidelik) moontlik. ‘n Diagnostiese algoritme wat gebaseer is op die
resultate van die HRS en sitologie-toetse, is voorgestel om pasiënte vroeër te behandel.
‘n Fluorometriese toets (FAST-Rif) is ontwikkel vir die vinnige diagnose van MDR-TB deur
mutasies in die rpoB-geen op te spoor met ‘n hoë sensitiwiteit en spesifisiteit (98% en 100%,
onderskeidelik). Hierdie mutasies is verantwoordelik vir weerstandigheid teen die antibiotikum
rifampicin (FAST-Rif) en word beskou as ‘n vinnige diagnose vir MDR-TB. Die FAST-Rif metode
kon maklik aangepas word om mutasies in die embB-gene, verantwoordelik vir weerstandigheid
teen die antibiotikum ethambutol (EMB), op te spoor. Die FAST-EMB-metode het ‘n sensitiwiteit
en spesifisiteit van 94.4% en 98.4% onderskeidelik getoon in vergelyking met DNS volgordebepaling.
Die FAST-EMB-metode was ‘n betekenisvolle verbetering op die onakkurate
kultuurgebaseerde metodes. ‘n Sterk korrelasie tussen EMB-weerstandigheid (en
weerstandigheid teen pyrazinamide) en MDR-TB is geïdentifiseer. Vervolgens is veranderinge
aan die Suid-Afrikaanse Nasionale TB-beheerprogram se Konsepbeleidsgids (2008) voorgestel.
Om die potensiële vrylating van amplikone te verhoed, is die FAST-Rif en FAST-EMB aangepas
tot ‘n enkelstap geslote buissisteem deur gebruik te maak van die opsporing van inhA promotormutasies
wat weerstandigheid teen isoniazid (INH) veroorsaak. Die metode het ‘n
sensitiwiteit en spesifisiteit van 100% en 83.3% onderskeidelik, getoon. Hierdie mutasies
veroorsaak laevlak weerstandigheid teen INH, maar ook kruisweerstandigheid teen ethionamide
(Eto). Aangesien daar ‘n sterk verbintenis tussen inhA-promotormutasies en XDR-TB in die Oos en
Wes-Kaapprovinsies van Suid-Afrika is, kan data van die GenoType® MDRTBPlus-toets
moontlik gebruik word om ‘n meer geïndividualiseerde behandeling te ontwerp afhangende van
die pasiënt se INH-mutasieprofiel. Ons behandelingsalgoritme is veral geskik vir XDR-TB pasiënte
vir wie daar weinig aktiewe antibiotika beskikbaar is.
Huidige diagnostiese metodes het almal voor- en nadele, dus bied ‘n kombinasie van fenotipiese
en genotipiese metodes moontlik die beste oplossing totdat beter metodes ontwikkel word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5479 |
| Date | 12 1900 |
| Creators | Hoek, Kim Gilberte Pauline |
| Contributors | Warren, Robin Mark, Van Helden, Paul David, Walzl, Gerhardt, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xiii, 174 p. : ill. |
| Rights | Stellenbosch University |
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