Temperature mediated heteroduplex analysis of rpoB mutations in Mycobacterium tuberculosis DNA: direct detection of rifampicin resistance in clinical specimens

Abstract number: 1134_01_160

Evans J., Parveen A., Boese K., Li X., Smith E., Hong G., Hawkey P.

Objectives:  

The frequency of resistance to rifampicin in some countries can be as high as 23%. Rifampicin and isoniazid are first-line drugs, and up to 90% of rifampicin resistant isolates are also isoniazid resistant, defined as Multi-Drug Resistant TB. (MDR-TB). 96% of rifampicin resistant isolates carry mutations in a 81-bp region of the rpoB gene. Therefore, it is of clinical importance to be able to rapidly detect rifampicin resistance in patients that are at risk of MDR-TB. Mutations in the rpoB gene of M. tuberculosis have been analysed by Temperature Mediated Heteroduplex Analysis (TMHA) previously, however, we make the first report of direct detection of rpoB mutations from clinical specimens.

Methods:  

100 rifampicin resistant isolates and 100 rifampicin sensitive isolates from the West Midlands, UK cultured in 2000–2004 had DNA extracted using the QIAamp DNA Mini-Kit. Primers were designed to amplify a 400 bp fragment of the M. tuberculosis rpoB gene that includes the 81-bp ‘hotspot’ region. Forward and reverse DNA sequencing defined mutations in the rpoB gene. From July 2004, all sputum samples that were rifampicin resistant frozen at –70°C and DNA was as above. An equal number of rifampicin sensitive sputum samples were also examined. To establish technique reproducibility each individual mutation was analysed in triplicate. After PCR and heteroduplexing, ìL of each PCR reaction was loaded onto a DNASep^® Cartridge in a WAVE^® DNA Fragment Analysis System (Transgenomic Inc., Omaha, NE). The gradient for separation was a 5.8-min gradient (400 bp) at a flow rate of 0.9 ml/min at 68°C.

Results:  

The 100 rifampicin resistant isolates possessed a range of defined rpoB mutations. The most common mutation was S531L, with H526Y being the second most prevalent. Mutations conferring rifampicin resistance in all of the 100 isolates were readily detected by the WAVE^® System. No false positive results were obtained when the 100 isolates with no resistance conferring mutations were analysed by dHPLC on the WAVE^® System. Mutation heteroduplexes were reproducible when analysed in triplicate on the WAVE^® System. All mutations defined in cultured isolates from the Midlands were detected directly in clinical sputum specimens.

Conclusion:  

Temperature mediated heteroduplex analysis of rpoB mutations conferring resistance to rifampicin in M. tuberculosis isolates and clinical specimens is an accurate, sensitive, cost-effective and reproducible technique.