Investigation into genotypic diagnostics for mycobacterium tuberculosis

[摘要] 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) andextensively drug resistant tuberculosis (XDR-TB). It is therefore essential that more rapiddiagnostic methods are developed. Molecular-based assays have the potential for the rapidspecies-specific diagnosis of TB and associated drug-resistances directly from clinicalspecimens. We investigated whether high resolution melting analysis (HRM) could enablethe rapid diagnosis of TB and associated drug resistance, since the HRM apparatus andreagents 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 lymphadenitisfrom fine-needle aspiration biopsy (FNAB) samples in 2 studies. This was done by targeting theregion of deletion 9 (RD9), present in M. tuberculosis and M. canettii, but absent from all othermembers 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 positiveculture). Despite this limitation our method was able to provide a rapid diagnosis in more thanhalf of the infected patients with a relatively high specificity (94.0% and 83.3%, respectively). Wetherefore proposed a diagnostic algorithm allowing the early treatment of patients with both HRMand cytology results indicative of mycobacterial disease.We developed the Fluorometric Assay for Susceptibility Testing of Rifampicin (FAST-Rif) whichallowed the rapid diagnosis of MDR-TB by detecting rifampicin (RIF) resistance mutations in therpoB gene with a sensitivity and specificity of 98% and 100%, respectively. The FAST-Rif methodwas easily adapted to detect ethambutol (EMB) resistance due to mutations in the embB genewith a sensitivity and specificity of 94.4% and 98.4% respectively, as compared to DNAsequencing. The FAST-EMB method was a significant improvement over the inaccurate culturebased method. We identified a strong association between EMB resistance (and pyrazinamideresistance) and MDR-TB and subsequently advised modifications to the current (2008) SouthAfrican National TB Control Programme draft policy guidelines.Due to the potential for amplicon release, we adapted the FAST-Rif and FAST-EMB methods toa closed-tube one-step method using the detection of inhA promoter mutations conferringisoniazid (INH) resistance as a model. The method (FASTest-inhA) was able to identify inhApromoter mutations with a sensitivity and specificity of 100% and 83.3%. These mutations are ofparticular 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 andEastern Cape Provinces of South Africa, data generated by the recently implementedGenoType® MDRTBPlus assay may allow individualised treatment regimens to be designed for apatient depending on their INH mutation profile. Our proposed treatment algorithm may beparticularly useful in XDR-TB cases, for which only few active drugs remain available.Since current diagnostic methods all carry advantages and disadvantages, a combination ofphenotypic and genotypic-based methodologies may be the best scenario while awaitingsuperior methods.

[发布机构] Stellenbosch University