1. Background Drug resistance continues to threaten tuberculosis (TB) control efforts despite the availability of rapid diagnostics and standardized regimens for treatment of multidrug-resistant (MDR) TB, which is resistant to rifampicin (RIF) and isoniazid (INH). Rapid and accurate drug susceptibility testing is crucial to ensure early initiation of appropriate therapy and delay in diagnosis increases the risk of patient mismanagement, the amplification of drug resistance and ongoing transmission. In 2019, an estimated 10 million people fell ill with TB; of those 10 million people, 3 million were not reported to have been diagnosed and notified. The gap is proportionately wider for drug-resistant TB (DR-TB). Of the estimated 465 000 patients with multidrug-resistant TB or RIF-resistant TB (MDR/RR-TB), only 206 030 (44%) were diagnosed and notified. For the first time, the World Health Organization (WHO) has provided global estimates of the incidence of INH resistance: in 2019, there were 1.4 million incident cases of INH-resistant TB, of which 1.1 million were susceptible to RIF. Most of these people were not diagnosed with DR-TB and did not receive appropriate treatment. 2. Molecular WHO-recommended rapid diagnostic tests (mWRDs) Sputum-smear microscopy is a relatively insensitive test and cannot distinguish drug-susceptible strains from drug-resistant strains. The current gold standard method for the bacteriological confirmation of TB is culture using commercially available liquid media. However, culture is not used as a primary diagnostic test in many high-burden countries because of the cost, the infrastructure requirements and the long time required to generate results Moreover, phenotypic DST remains the reference standard for most anti-TB compounds; however, this test is slow and it requires specialized infrastructure and highly skilled staff. 1) Xpert MTB/RIF The Xpert MTB/RIF assay is a cartridge-based automated test that uses real-time polymerase chain reaction (PCR) on the GeneXpert platform to identify MTBC and mutations associated with RIF resistance directly from sputum specimens in less than 2 hours. WHO recommends using the Xpert MTB/RIF as an initial test for TB and detection of RIF resistance in adults and children with signs and symptoms of pulmonary TB. In addition, Xpert MTB/RIF can be used for TB meningitis and extrapulmonary TB. The Xpert MTB/RIF Ultra assay (hereafter called Xpert Ultra) uses the same GeneXpert platform as the Xpert MTB/RIF test and was developed to improve the sensitivity and reliability of detection of MTBC and RIF resistance. To address sensitivity, Xpert Ultra uses two multicopy amplification targets (IS6110 and IS1081) and a larger PCR chamber; thus, Xpert Ultra has a lower LoD than Xpert MTB/RIF (16 colony forming units [cfu]/mL and 131 cfu/mL, respectively). At very low bacterial loads, Xpert Ultra can give a “trace” result, which is not based on amplification of the rpoB target and therefore does not give results for RIF susceptibility or resistance. An additional improvement in the Xpert Ultra is that the analysis is based on melting temperature (Tm), which allows for better differentiation of resistance-conferring mutations. Planning the transition to the Xpert Ultra requires special consideration and a GLI document is available to assist in this process. 2) Moderate Complexity automated NAATs Rapid detection of TB and RIF resistance is increasingly available as new technologies are developed and adopted by countries. However, what has also emerged is the relatively high burden of INH-resistant, RIF-susceptible TB that is often undiagnosed. Globally, INH-resistant, RIF-susceptible TB is estimated to occur in 13.1% of new cases and 17.4% of previously treated cases. A new class of technologies has come to market with the potential to address this gap. Several manufacturers have developed moderate complexity automated NAATs for detection of TB and resistance to RIF and INH on high throughput platforms for use in laboratories. The tests belonging to this class are faster and less complex to perform than phenotypic culture based drug susceptibility testing (DST) and line-probe assays (LPA). They have the advantage of being largely automated following the sample preparation step. Moderate complexity automated NAATs may be used as an initial test for detection of TB and resistance to both first-line TB drugs simultaneously (RIF and INH). They offer the potential for the rapid provision of accurate results and for testing efficiency where high volumes of tests are required daily. Hence, these technologies are suited to areas with a high population density and rapid sample referral systems. WHO convened a Technical Expert Group to assess the results of an external laboratory validation of four novel centralized TB assays: the Abbott RealTime MTB and MTB RIF/INH assays, the Roche cobasR MTB and MTB-RIF/INH assays, the Hain FluoroTypeR MTBDR assay, and the BD MAXTM MDR-TB assay). The moderate complexity automated NAATs class of tests includes rapid and accurate tests for the detection of pulmonary TB from respiratory samples. Overall pooled sensitivity for TB detection was 93.0% and specificity 97.7%. Moderate complexity automated NAATs are also able to simultaneously detect resistance to both RIF and INH, and are less complex to perform than phenotypic DST and LPAs. After the sample preparation step, the tests are largely automated. Overall pooled sensitivity for detection of RIF resistance was 96.7% and specificity was 98.9%. Table 1. Mycobacterium genomic regions targeted by different assays for TB detection