Rapid diagnosis of MDR and XDR tuberculosis with the MeltPro TB assay in China

Despite encouraging progress in tuberculosis (TB) control, TB remains a leading cause of morbidity and mortality worldwide1,2. The World Health Organization (WHO) estimated that there were 9 million incident TB cases and 1.5 million deaths from TB in 20133. The emergence of drug-resistant TB—especially multidrug-resistant TB (MDR-TB, defined as resistance to at least isoniazid and rifampicin) and extensively drug-resistant TB (XDR-TB, defined as MDR-TB plus resistance to any fluoroquinolone and kanamycin, amikacin, or capreomycin)—is considered the greatest obstacle to global TB control due to difficulties in diagnosis and treatment4,5,6. Globally, in 2013, an estimated 480,000 and 43,200 people developed MDR-TB and XDR-TB, respectively. However, only 136,000 MDR-TB cases (28%) were detected and notified, and the situation for detecting XDR-TB was even more unsatisfactory because of the lack of capability to detect susceptibility against second-line drugs in many TB high-burden countries3. Hence, there is an urgent need to ensure that all drug-resistant TB suspects undergo testing for susceptibility to anti-TB drugs to initiate effective treatment regimens and appropriate measures of infection control2,7. Because of the slow growth rate of Mycobacterium tuberculosis (MTB), conventional phenotypic drug susceptibility testing (DST) typically takes 1 to 3 months to determine the drug resistance profiles of MTB isolates8,9,10. More importantly, all culture-based conventional methods require a biosafety category 3 laboratory facility and extensive training of personnel, requirements that are largely unattainable in developing countries10. In recent years, several commercial molecular tests have been developed to determine the drug resistance of MTB isolates based on the detection of specific genetic mutations conferring resistance2,11,12,13,14. Of these rapid tests, the GenoType MTBDR (Hain Lifescience, Germany) and GeneXpert (Cepheid, USA) have been endorsed by WHO for the detection of rifampicin (RIF) and/or isoniazid (INH) resistance8. Although most available molecular diagnostics detect resistance to RIF and INH, the choice of molecular assays to detect resistance to second-line drugs is far more limited, except for GenoType MTBDRsl from Hain Lifescience7. On the basis of published reports, WHO decided not to recommend this assay as a replacement for conventional DST to rule out resistance in routine clinical practice15. The shortage of DST technologies for second-line drugs highlights the need for the development and evaluation of new molecular tools to improve diagnosis of MDR- and XDR-TB. The MeltPro TB assay, developed by Zeesan Biotecheh (Xiamen, China), is an innovative molecular test for detection of resistance to the main first-line and second-line anti-TB drugs16. Unlike commercially available molecular tests, this technology is based on melting curve analysis with dually labeled probes, which retrieves the melting temperature (Tm) shift from the wide-type into the genetic mutation of MTB. The intrinsic feature of this assay makes it possible to cover two fragments (more than 20 continuous nucleotides per fragment) associated with drug resistance in one assay; it is also easy to perform without cumbersome hybridization16,17. The MeltPro TB assays for detecting RIF, INH and fluoroquinolone resistance have been officially approved by the China Food and Drug Administration (CFDA), marking them possible for clinical practice. Several previous studies found that the sensitivity and specificity of MeltPro were 93.4% and 97.4%, respectively, for RIF resistance, and 90.8% and 96.4%, respectively, for INH resistance using the proportion method on the solid medium as a reference standard16,18, suggesting that this assay could be used as an alternative to phenotypic DST. However, these studies evaluated the MeltPro TB assay on cultured isolates, and assay performance for detecting resistance to second-line drugs was not evaluated. Thus, there is a need to validate assay performance in settings with a high prevalence to evaluate the feasibility of using the test for routine detection of XDR-TB cases from clinical samples. In this paper, we report on a multicenter study to evaluate use of the MeltPro TB assay on sputum samples for detection of resistance to first-line and second-line TB drugs. The results provide insight on the potential to scale up this new technology in China.