Targeted NGS tests couple amplification of selected genes with NGS technology to detect resistance to many drugs with a single test. Because targeted NGS tests can interrogate entire genes to identify specific mutations associated with resistance, targeted NGS tests may provide improved accuracy compared with other WRDs. In addition, new targeted NGS tests can detect resistance to new and repurposed drugs not currently included in any other molecular assays. Thus, targeted NGS tests offer great potential to provide comprehensive resistance detection matched to modern treatment regimens (21).
Fig. 2.2. Process of testing, from sample to result report, for targeted NGS
The class of targeted NGS tests was defined as tests that use massively parallel sequencing to detect resistance to TB drugs, starting from a processed clinical sample and ending with an end-user report that relates detected M. tuberculosis mutations to the presence (or absence) of drug resistance, based on the interpretation of a standard catalogue of mutations (Fig. 2.2). The first-in-class products include Deeplex® Myc-TB (GenoScreen) for RIF, INH, PZA, EMB, FQ, BDQ, LZD, CFZ, AMK and STR; AmPORE TB (Oxford Nanopore Technologies) for RIF, INH, FQ, LZD, AMK and STR; and TBseq® (ShengTing Biotech) for EMB. Where a product has not yet met the requirements for a specific drug (i.e. the drug is not currently listed), further improvements to the product and a review of the evidence will be necessary before that drug can be put into clinical use.
Among people with bacteriologically confirmed pulmonary TB, the targeted NGS test performances (Table 3.5 in Section 3) were determined to be accurate for all drugs included in the assessment, with pooled sensitivity of at least 95% for INH, moxifloxacin (MFX) and EMB; more than 93% for RIF and levofloxacin (LFX); and 88% for PZA. The pooled specificity was at least 96% for all drugs. The reference standard was phenotypic DST for INH, LFX and MFX, and a combination of phenotypic DST and whole-genome sequencing (WGS) for RIF, PZA and EMB. The indeterminate rate ranged from 9% (LFX and MFX) to 18% (PZA), but was highest in samples with low or very low bacterial loads, which may have implications for implementation; therefore, priority should be given to samples with a higher bacillary load. The overall certainty of the evidence ranged between low and moderate for test accuracy.
Among people with bacteriologically confirmed RIF-resistant pulmonary TB, the targeted NGS test performances (Table 3.6 in Section 3) were determined to be accurate for INH, LFX, MFX, STR and EMB (pooled sensitivity ≥95%), and acceptable for BDQ (68%), LZD (69%), CFZ (70%), AMK (87%) and PZA (90%). The specificity was at least 95% for all drugs except STR (75%). The reference standard was phenotypic DST for all drugs except EMB and PZA, where a combination of phenotypic DST and WGS was used. The indeterminate rate ranged from 9% (LFX and MFX) to 21% (EMB), and depended on the bacterial load. For test accuracy, the overall certainty of the evidence ranged from low to high.
WHO recommends the use of targeted NGS tests as follow-on tests to detect drug resistance in the following situations (7):
- In people with bacteriologically confirmed pulmonary TB disease, targeted NGS tests may be used on respiratory samples to diagnose resistance to RIF, INH, FQ, PZA and EMB rather than phenotypic DST.
- In people with bacteriologically confirmed RIF-resistant pulmonary TB disease, targeted NGS tests may be used on respiratory samples to diagnose resistance to INH, FQ, BDQ, LZD, CFZ, PZA, EMB, AMK and STR rather than phenotypic DST.
Notes:
- The targeted NGS tests do not replace existing rapid tests that are more accessible and easier to perform for detecting resistance to RIF, INH and FQ. However, if targeted NGS tests can be performed rapidly, they can be considered as an alternative initial option for prioritized populations. Those who will benefit most from these tests are individuals requiring rapid and comprehensive DST, where there is limited access to phenotypic DST.
- The usefulness of the targeted NGS tests depends on the information provided in the WHO catalogue of mutations (4), which allows for interpretation of resistance data. Regular updating of that catalogue – incorporating additional genetic targets, including new drugs (e.g. Pa) – is needed to enhance the sensitivity and specificity of targeted NGS tests.
- An implementation consideration is that the indeterminate rate of the currently available targeted NGS tests ranged from 9% to 21%, and was higher in samples with low bacillary load. Priority should be given to testing samples with a high bacillary load, as determined by initial bacteriological tests (e.g. semiquantitative grading: high or medium, or smear-positive). In situations where the bacillary load is low (e.g. semiquantitative grading: low, very low or trace, or smear-negative), the recommendations still hold, while acknowledging the higher rates of indeterminate results. Annex 3 provides additional resources guiding the implementation of targeted NGS.