Interpretation of discordant results
This algorithm follows from Algorithm 1 with an mWRD that detected MTBC and was RIF susceptible. In the scenario where the moderate complexity automated NAAT was used, the INH result would already be available. In this algorithm, the follow-up testing could have a second RIF result when the FL-LPA is used as a follow-on, or it could have a second INH result when the moderate complexity automated NAAT is followed by the low complexity automated NAAT. Sometimes, results may be discordant. Each discordant result will need to be investigated on a case-by-case basis. General considerations are outlined below.
- Where the mWRD (e.g. Xpert Ultra) result is “MTBC detected”, and the follow-on FL-LPA result is “MTB not detected” or “uninterpretable”:
- mWRDs recommended for detection of TB have a lower LoD than the FL-LPA; thus, FL-LPA may fail to detect TB in mWRD-positive samples that contain few bacilli. For example, it is estimated that only about 80% of specimens with “MTBC detected” by Xpert MTB/RIF will generate an FL-LPA result that can be interpreted.
- The initial mWRD result should be used to guide treatment decisions, pending additional testing
- Follow-up actions may include submitting a specimen for culture and a molecular or phenotypic testing of the recovered isolate, and evaluating the possibility of laboratory or clerical error.
- Where the initial mWRD result is “MTBC detected, RIF resistance not detected” and the sample is RIF resistant by FL-LPA:
- Treatment decisions should be based on the FL-LPA result (i.e. based on the worst-case scenario).
- This result is expected to be rare because both assays interrogate the same region of the rpoB gene. There have been reports of mWRD RIF-susceptible and FL-LPA RIF-resistant discordances, but data are too sparse to assess how frequently this occurs.
- FL-LPA is more sensitive for identifying RIF resistance than most mWRDs in heteroresistant populations (i.e. mixtures of susceptible and resistant bacteria). The test includes hybridization probes specific to both the common mutated and the wild-type sequences in the bacterial genome. If the Xpert Ultra is used, a review of the probe melting temperature curves may be helpful to identify heteroresistant populations (e.g. dual peak).
- Follow-up actions may include DNA sequencing, conducting phenotypic DST, and evaluating the possibility of laboratory or clerical error.
- Where the moderate complexity automated NAAT result is “MTBC detected, RIF resistance not detected, INH resistance detected” but the result is “INH susceptible” by low complexity automated NAAT:
- This result is expected to be rare because both assays interrogate the same region of the katG and inhA genes.
- The existence of heteroresistant populations (i.e. mixtures of susceptible and resistant bacteria) is a more likely reason, especially in high-burden settings where the force of infection is high. A review of the low complexity automated NAAT probe melting temperatures (52) may identify such a possibility (e.g. dual peak).
- Follow-up actions may include DNA sequencing, conducting phenotypic DST, and evaluating the possibility of laboratory or clerical error.
- Reassess the risk for Hr-TB, and if a high risk for Hr-TB or administration errors (e.g. mislabelling) are not the reason, treatment decisions should cover the worst-case scenario and be based on the moderate complexity automated NAAT result.
- Where the moderate complexity automated NAAT result is “MTBC detected, RIF resistance not detected, INH resistance not detected” and the sample is INH resistant by low complexity automated NAAT:
- Treatment decisions should be based on the low complexity automated NAAT result (i.e. treat based on the worst-case scenario).
- This result is expected to be rare because both assays interrogate the same region of the katG and inhA genes. However, the low complexity automated NAAT is more sensitive for INH detection because it includes additional gene targets (fabG1 and oxyR-ahpC intergenic regions).
- The existence of heteroresistant populations (i.e. mixtures of susceptible and resistant bacteria) is another possible reason, especially in high-transmission settings. A review of the low complexity automated NAAT probe melting temperatures may identify such a possibility (e.g. dual peak).
- Follow-up actions may include DNA sequencing, conducting phenotypic DST, and evaluating the possibility of laboratory or clerical error.
- Targeted NGS detects resistance and other molecular tests show susceptibility or vice versa:
- Check to see whether the mutation detected by targeted NGS was in a region not covered by the other molecular test. If that is the case, the targeted NGS should be taken as the final result.
- Check to see whether the mutation is a synonymous mutation by targeted NGS. If that is the case, it would indicate that the other molecular test result is incorrect.
- Check to see whether targeted NGS detected heteroresistance. If that is the case, the most resistant profile should be used for clinical management. Targeted NGS is better at resolving heteroresistance than other molecular tests.