Genotypic (molecular) methods have considerable advantages for scaling up programmatic management and surveillance of drug-resistant TB, offering rapid diagnosis, standardized testing, potential for high throughput and fewer requirements for laboratory biosafety. Molecular tests for detecting drug resistance – for example, the GenoType MTBDRsl assay (Hain Lifescience, Nehren, Germany), hereafter referred to as MTBDRsl (17) – have shown promise for the diagnosis of drug-resistant TB. These tests are rapid (can be performed in a single working day) and detect the presence of mutations associated with drug resistance. MTBDRsl belongs to a category of molecular genetic tests called second-line LPAs (SL-LPAs).
MTBDRsl (version 1.0) was the first commercial SL-LPA for detection of resistance to second-line TB drugs. In 2015, the manufacturer developed and made commercially available version 2.0 of the MTBDRsl assay. Version 2.0 detects the mutations associated with fluoroquinolones and second-line injectable drug (SLID) resistance detected by version 1.0, and additional mutations. Once a diagnosis of MDR/RR-TB has been established, an SL-LPA can be used to detect additional resistance to second-line drugs.
The MTBDRsl assay incorporates probes to detect mutations within genes that are associated with resistance to either fluoroquinolones or SLIDs (gyrA and rrs for version 1.0 and those genes plus gyrB and the eis promoter for version 2.0). The presence of mutations in these regions does not necessarily imply resistance to all the drugs within a particular class. Although specific mutations within these regions may be associated with different levels of resistance (i.e. different minimum inhibitory concentrations) to each drug within these classes, the extent of cross-resistance is not completely understood.
1. For patients with confirmed MDR/RR-TB, SL-LPA may be used as the initial test, instead of phenotypic culture-based DST, to detect resistance to fluoroquinolones.2.
2. For patients with confirmed MDR/RR-TB, SL-LPA may be used as the initial test, instead of phenotypic culture-based DST, to detect resistance to the SLIDs.
- These recommendations apply to the use of SL-LPA for testing sputum specimens (direct testing) and cultured isolates of M. tuberculosis (indirect testing) from both pulmonary and extrapulmonary sites. Direct testing on sputum specimens allows for the earlier initiation of appropriate treatment
- These recommendations apply to the direct testing of sputum specimens from MDR/RR-TB, irrespective of the smear status, while acknowledging that the indeterminate rate is higher when testing smear-negative sputum specimens than with smear-positive sputum specimens.
- These recommendations do not eliminate the need for conventional phenotypic DST capacity, which will be necessary to confirm resistance to other drugs and to monitor the emergence of additional drug resistance.
- Conventional phenotypic DST can still be used in the evaluation of patients with negative SL-LPA results, particularly in populations with a high pretest probability for resistance to fluoroquinolones or SLID (or both).
- These recommendations apply to the use of SL-LPA in children with confirmed MDR/RR-TB, based on the generalization of data from adults.
- Resistance-conferring mutations detected by SL-LPA are highly correlated with phenotypic resistance to ofloxacin and levofloxacin.
- Resistance-conferring mutations detected by SL-LPA are highly correlated with phenotypic resistance to SLID.
- Given the high specificity for detecting resistance to fluoroquinolones and SLID, the positive results of SL-LPA could be used to guide the implementation of appropriate infection control precautions.
The SL-LPA is based on the same principle as the first-line LPA. The assay procedure can be performed directly using a processed sputum sample or indirectly using DNA isolated and amplified from a culture of M. tuberculosis. Direct testing involves the following steps:
1) Decontamination (e.g. with sodium hydroxide) and concentration of a sputum specimen by centrifugation.
2) Isolation and amplification of DNA.
3) Detection of the amplification products by reverse hybridization.
4) Visualization using a streptavidin-conjugated alkaline phosphatase colour reaction.
Indirect testing includes only Steps 2–4. The observed bands, each corresponding to a wild-type or resistance-genotype probe, can be used to determine the drug susceptibility profile of the analysed specimen. The assay can be performed and completed within a single working day.
The index test used was MTBDRsl, and the different characteristics of versions 1.0 and 2.0 are presented in Table 2.3.3. SL-LPAs detect specific mutations associated with resistance to the class of fluoroquinolones (including ofloxacin, levofloxacin, moxifloxacin and gatifloxacin) and SLIDs (including kanamycin, amikacin and capreomycin) in the MTBC. Version 1.0 detects mutations in the gyrA quinolone resistance-determining region (codons 85–97) and rrs (codons 1401, 1402 and 1484). Version 2.0 additionally detects mutations in the gyrB quinolone resistance-determining region (codons 536–541) and the eis promoter region (codons –10 to –14) (40). Mutations in these regions may cause additional resistance to the fluoroquinolones or SLIDs, respectively; thus, version 2.0 is expected to have improved sensitivity for resistance to these drug classes. Mutations in some regions (e.g. the eis promoter region) may be responsible for causing resistance to one drug in a class more than other drugs within that class. For example, the eis C14T mutation is associated with kanamycin resistance in strains from Eastern Europe (41). Version 1.0 also detects mutations in embB that may encode for resistance to ethambutol. Because ethambutol is a first-line drug and was omitted from version 2.0, this review did not determine the accuracy for ethambutol resistance.
More data are needed to better understand the correlation of the presence of certain fluoroquinolone resistance-conferring mutations with phenotypic DST resistance and with patient outcomes.
Fig. 2.3.6 shows an example of MTBDRsl results for version 1.0 and 2.0. A band for the detection of the MTBC (the "TUB" band) is included, as well as two internal controls (conjugate and amplification controls), and a control for each gene locus (version 2.0: gyrA, gyrB, rrs, eis). The two internal controls plus each gene locus control should be positive, otherwise the assay cannot be evaluated for that particular drug. A result can be indeterminate for one locus but valid for another (on the basis of a gene-specific locus control failing).
A template is supplied by the manufacturer to help the user to read the strips where the banding patterns are scored by eye, transcribed and reported. In high-volume settings, the GenoScan®, an automated reader, can be incorporated to interpret the banding patterns automatically and give a suggested interpretation. If the operator agrees with the interpretation, the results are automatically uploaded, thereby reducing possible transcription errors.
Justification and evidence
In March 2016, WHO’s Global TB Programme convened a GDG to assess available data on the use of the MTBDRsl assay. WHO commissioned a systematic review on the accuracy and clinical use of assays for the detection of mutations associated with resistance to fluoroquinolones and SLID in people with MDR/RR-TB.
The PICO questions in Box 2.3.3 were designed to form the basis for the evidence search, retrieval and analysis.
Twenty-nine unique studies were identified; of these, 26 evaluated the MTBDRsl version 1.0 assay (including 21 studies from the original Cochrane review). Three studies (one published and two unpublished) evaluated version 2.0. Data for version 1.0 and version 2.0 of the MTBDRsl assay were analysed separately. A phenotypic culture-based DST reference standard was used for the primary analyses. These analyses were stratified first by susceptibility or resistance to a particular drug, and second by type of SL-LPA testing (indirect testing or direct testing).
Performance of SL-LPA on sputum specimens and culture isolates
In patients with MDR/RR-TB, a positive SL-LPA result for fluoroquinolone resistance (as a class) or SLID resistance (as a group) can be treated with confidence. The diagnostic accuracy of SL-LPA is similar when performed directly on sputum specimens or indirectly on cultured isolates of M. tuberculosis.
Given the confidence in a positive result and the ability of the test to provide rapid results, the GDG felt that SL-LPA may be considered for use as an initial test for resistance to the fluoroquinolones and when relevant SLIDs. However, when the test shows a negative result, phenotypic culture-based DST may be necessary, especially in settings with a high pretest probability for resistance to either fluoroquinolones or SLIDs (or both). The use of SL-LPA in routine care should improve the time to the diagnosis of fluoroquinolone and where relevant SLIDs, especially when used for the direct testing of sputum specimens of patients with confirmed MDR/RR-TB. Early detection of drug resistance should allow for the earlier initiation of appropriate patient therapy and improved patient health outcomes. Overall, the test performs well in the direct testing of sputum specimens from patients with confirmed MDR/RR-TB, although the indeterminate rate is higher when testing smear-negative sputum specimens compared with smear-positive sputum specimens.
When the MTBDRsl assay is used in the direct testing of smear-negative sputum specimens from a population of patients with confirmed drug-resistant TB, up to 44% of the results may be indeterminate (less with version 2.0, although very limited data) and hence require repeat or additional testing. However, if the same test were to be applied to the testing of smear-negative sputum specimens from patients without confirmed TB or drug-resistant TB (i.e. patients suspected of having drug-resistant TB), the indeterminate rate for the test would be significantly higher. Given the test’s sensitivity and specificity when an SL-LPA is done directly on sputum, the GDG felt that SL-LPAs can be used for the testing of all sputum specimens from patients with confirmed MDR/RR-TB, irrespective of whether the microscopy result is positive or negative.
For the reasons mentioned above (inadequate data owing to too few studies on version 2.0), results are not presented here for version 2.0. For MTBDRsl version 2.0, the data were either too sparse or too heterogeneous to combine in a meta-analysis or to compare indirect and direct testing.
Three studies evaluated the MTBDRsl version 2.0 in 562 individuals, including 111 confirmed cases of TB with fluoroquinolone resistance by indirect testing on a culture of M. tuberculosis compared with a phenotypic culture-based DST reference standard. Estimates of sensitivity ranged from 84% to 100% and specificity from 99% to 100%.
See Web Annex 4.6 "Drug concentrations used in culture-based DST SL-LPA" for details of the drug concentrations used in culture-based DST to evaluate the performance of SL-LPAs in each included study.
The SL-LPA should only be used to test specimens from patients with confirmed MDR/RR-TB. Adoption of SL-LPAs does not eliminate the need for conventional culture and DST capability. Despite good specificity of SL-LPAs for the detection of resistance to fluoroquinolones and the SLIDs, culture and phenotypic DST is required to completely exclude resistance to these drug classes as well as to other second-line drugs. The following implementation considerations apply:
- SL-LPAs cannot determine resistance to individual drugs in the class of fluoroquinolones. Resistance-conferring mutations detected by SL-LPAs are highly correlated with phenotypic resistance to ofloxacin and levofloxacin.
- Mutations in some regions (e.g. the eis promoter region) may be responsible for causing resistance to one drug in a class more than other drugs within that class. For example, the eis C14T mutation is associated with kanamycin resistance in strains from Eastern Europe.
- SL-LPAs should be used in the direct testing of sputum specimens, irrespective of whether samples are smear negative or smear positive.
- SL-LPAs are designed to detect TB and resistance to fluroquinolones and SLIDs from sputum samples. Other respiratory samples (e.g. bronchoalveolar lavage and gastric aspirates) or extrapulmonary samples (tissue samples, CSF or other body fluids) have not been adequately evaluated.
- Culture and phenotypic DST plays a critical role in the monitoring of a patient's response to treatment, and in detecting additional resistance to second-line drugs during treatment.
- SL-LPAs are suitable for use at the central or national reference laboratory level; they can also be used at the regional level if the appropriate infrastructure can be ensured (three separate rooms are required).
- All patients identified by SL-LPAs should have access to appropriate treatment and ancillary medications.
- Development of improved understanding of the correlation between the detection of resistance-conferring mutations with phenotypic DST results and with patient outcomes.
- Development of improved knowledge of the presence of specific mutations detected with SL-LPA correlated with minimum inhibitory concentrations for individual drugs within the classes of fluoroquinolones and SLIDs.
- Determination of the limit of detection of SL-LPA for the detection of heteroresistance.
- Gathering of more evidence on the impact of MTBDRsl on appropriate MDR-TB treatment initiation and mortality.
- Strongly encourage that future studies follow the recommendations in the STARD (42) statement to improve the quality of reporting.
- Performance of country-specific cost-effectiveness and cost-benefit analyses of the use of SL-LPA in different programmatic settings.