Rapid Tuberculosis diagnostics including molecular first- and second-line resistance testing based on a novel microfluidic DNA extraction cartridge.

Abstract Xpert MTB/RIF testing (Xpert, Cepheid, Sunnyvale, CA) has improved tuberculosis (TB) diagnostics and rifampicin (Rif) resistance testing world-wide. However, it has weaknesses such as its restriction to Rif resistance testing and that the extracted DNA cannot be used for further testing. Here, a holistic diagnostic workflow including TB detection as well as genetic resistance testing towards Rif, isoniazid (Inh) and important second-line drugs (SLDs) based on a novel microfluidic DNA-extraction cartridge (TB-Disk) is presented. DNA from 73 pre-characterized sputum samples was extracted with TB-Disk including 45 clinical and bacteriologically confirmed TB samples, nine TB negative samples and 19 sputum samples spiked with two-fold dilutions of TB bacteria. The extracted DNA was subjected to further testing with FluoroType MTB (FT-MTB), GenoType MTBDRplus (GT-plus) and GenoType MTBDRsl (GT-sl, Hain Lifescience, Nehren, Germany). 100% (20/20) and 72% (18/25) of smear-positive and smear-negative TB samples were identified as MTBC-positive. 79% (33/42) of subsequently GT-plus tested samples yielded a valid result. Eight samples were identified as MDR-TB by GT-plus and further tested for resistance towards SLDs using GT-sl yielding 75% (6/8) valid results. FT-MTB with Disk-DNA and DNA extracted with FluoroLyse (FL-DNA) yielded similar analytical sensitivities. FT-MTB with Disk-DNA was 100% specific. TB-Disk in combination with FT-MTB allows for highly sensitive TB detection. The Disk-DNA can be further used for screening resistance towards first- and second-line drugs.