Treatment of TB has undergone significant changes over recent years, with new drugs and regimens recommended; hence, the definitions for DR-TB have been revised accordingly. The updated pre-XDR-TB definition is "TB caused by M. tuberculosis strains that fulfil the definition of MDR/RR-TB and are also resistant to any FQ". The updated XDR-TB definition is "TB caused by M. tuberculosis strains that fulfil the definition of MDR/RR-TB and that are also resistant to any FQ and at least one additional Group A drug (i.e. BDQ or LZD)" (18).²⁰ These changes have important implications for Member States, particularly for scaling up the detection of resistance to FQ and BDQ. In addition, there is an increasing demand for DST for other new and repurposed drugs. In 2018, WHO updated the critical concentrations (CCs) used in phenotypic DST to include the new and repurposed drugs for existing methods (19).²¹ A WHO technical manual for DST is also available (20).²²
The new definition of XDR-TB requires DST results for FQs, BDQ and LZD; thus, testing for resistance to BDQ and LZD has become a priority, particularly testing for BDQ resistance. Culture-based phenotypic DST for BDQ and LZD can be performed using either the mycobacterial growth indicator tube (MGIT) or Middlebrook 7H11 media. The BDQ pure drug substance for use in phenotypic DST is provided free through the US National Institutes of Health (NIH) HIV Reagent Program (21);²³ however, courier costs need to be covered. An information note that explains the request process is also available (22).²⁴ Delamanid powder can be ordered from the manufacturer, Otsuka, through the website of the American Type Culture Collection (ATCC) (delamanid: NR-51636) (23).²⁵ Other pure drug substances for laboratory DST and associated catalogues are available through the Global Drug Facility (24).²⁶
In addition, CCs for the rifamycins and INH were reviewed, and findings published in 2021 (25).²⁷ The INH CCs have remained the same, whereas the RIF CCs in MGIT and 7H10 have been revised downwards from 1.0 mg/L to 0.5 mg/L. These revisions are expected to reduce discordance between genotypic and phenotypic DST results, and laboratories are advised to implement these changes immediately. For MGIT, this can be achieved by reconstituting the lyophilized RIF from the BD SIRE kit in 8 mL, instead of 4 mL, of sterile distilled or deionized water.
The latest CCs for all drugs are listed in Table 2.2 (22).²⁸
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. New and rapid next-generation technologies are needed at the peripheral level to expedite appropriate therapy and impact patients' outcomes. WHO has developed a target product profile, planned for release in the second half of 2021, to guide research and development to address this need.³²
DNA sequencing using next-generation sequencing (NGS) is a promising method for rapid detection of mutations associated with drug resistance for many anti-TB drugs (28).³³ NGS-based DST could reduce the need for phenotypic DST for patient-care decisions and DR-TB surveys, and it may be particularly useful for drugs for which phenotypic DST is unreliable or settings that do not have the capacity to perform phenotypic DST. The current NGS systems have limitations, especially the required computational expertise and resources. Thus, implementation of NGS-based DST is likely to be focused, at least initially, on capacity-building at the central level (i.e. national TB reference laboratory [NTRL] and well-performing regional TB reference laboratories).
Amplification-based targeted NGS assays for detecting DR-TB directly from sputum specimens have been developed. The Deeplex-MycTB assay (29)³⁴ (GenoScreen, Lille, France) is commercially available (CE-IVD and RUO versions available). The assay allows prediction of resistance to 15 anti-TB drugs, with a turnaround time of less than 48 hours. Similarly, the Translational Genomics Research Institute (Phoenix, Arizona, USA) is currently developing a next-generation rapid DST assay that can detect mutations associated with resistance to at least seven drugs (30).³⁵ Also, through ABL it has commercialized an assay that allows detection of resistance to RIF and INH (i.e. DeepChek-TB RpoB/InhA Genotyping Assay) (31).³⁶ Furthermore, a new product, the NanoTB Drug Resistance Assay (Oxford Nanopore Technologies, Oxford, United Kingdom of Great Britain and Northern Ireland), can detect resistance to 11 drugs and has the advantage of working on small devices such as the MinION, a handheld compact device (32).³⁷ These assays have not yet been reviewed or approved by WHO for clinical use.
One important issue identified when using these technologies is the lack of a standardized and robust single reference source for interpretation of mutations. To address this need, WHO has developed guidance (33)³⁸ and has released a catalogue of mutations in MTBC and their association with resistance. This is the largest collated database of MTBC isolates (>38 000) with matched phenotypic DST and whole genome sequences. The methods used to generate the catalogue and findings are presented in the report that accompanies the full catalogue, which is now available.³⁹ Future regular updates are planned. The catalogue is also expected to support the development of other rapid molecular DST methods.
²⁰ See https://www.who.int/publications/i/item/meeting-report-of-the-who-expert-consultation-on-the-definition-ofextensively-drug-resistant-tuberculosis
²¹ See https://www.who.int/publications/i/item/WHO_CDS_TB_2018.5
²² See https://www.who.int/publications/i/item/9789241514842
²³ See https://www.hivreagentprogram.org/
²⁴ See http://stoptb.org/assets/documents/resources/publications/sd/BDQ_DEL_access.pdf
²⁵ See https://www.atcc.org
²⁶ See http://www.stoptb.org/gdf/drugsupply/product_catalog.asp
²⁷ See https://www.who.int/publications/i/item/technical-report-on-critical-concentrations-for-drugsusceptibility-testing-of-isoniazid-and-therifamycins-%28rifampicin-rifabutin-and-rifapentine%29
²⁸ See http://stoptb.org/assets/documents/resources/publications/sd/BDQ_DEL_access.pdf
²⁹ See https://www.who.int/publications/i/item/technical-report-on-critical-concentrations-for-drugsusceptibility-testing-ofisoniazid-and-therifamycins-%28rifampicin-rifabutin-and-rifapentine%29
³⁰ See http://www.stoptb.org/wg/gli/assets/documents/Updated%20critical%20concentration%20table_1st%20and%20 2nd%20line%20drugs.pdf
³¹ See http://apps.who.int/iris/bitstream/10665/260470/1/WHO-CDS-TB-2018.5-eng.pdf
³² The document will be titled WHO target product profiles for next-generation drug susceptibility tests at peripheral level.
³³ See https://apps.who.int/iris/bitstream/handle/10665/274443/WHO-CDS-TB-2018.19-eng.pdf
³⁴ See https://www.genoscreen.fr/en/genoscreen-services/products/deeplex
³⁵ See https://www.tgen.org/news/2019/march/18/tgen-tb-test-to-help-eliminate-disease-worldwide/
³⁶ See https://www.ablsa.com/laboratory-applications/deepchek-hbv-rt-genotyping-dr-assay-2-2/
³⁷ See https://nanoporetech.com/resource-centre/rapid-diagnosis-drug-resistant-tuberculosis-using-nanopore-sequencing
³⁸ See https://www.who.int/publications/i/item/WHO-CDS-TB-2018.19
³⁹ Catalogue of mutations in Mycobacterium tuberculosis complex and associated drug resistance.
CB: clinical breakpoint; CC: critical concentration; DST: drug-susceptibility testing; LJ: Löwenstein–Jensen; MGIT: mycobacterial growth indicator tube; RFB: rifabutin; RIF: rifampicin; RPT: rifapentine; TB: tuberculosis; WHO: World Health Organization
Note: All concentrations are in mg/L and apply to the proportion method, with 1% as the critical proportion. Unless otherwise stated, they are CCs rather than CBs. Red font indicates updated CC for RIF in 2021.
ᵃ MGIT is proposed as the reference method for performing DST for second-line anti-TB medicines.
ᵇ Additional data are needed to clarify whether the RIF CC for LJ is set correctly. The RIF CC for 7H11 was based on limited data and might be too high given that the RIF CC for 7H10 had to be lowered to 0.5 mg/L.
ᶜ No CCs were adopted because RFB is not currently recommended for TB treatment by WHO, but the validity of the current CCs of 0.5 mg/L for 7H10, 7H11 and MGIT set by the Clinical and Laboratory Standards Institute could not be confirmed by WHO (27). The conservative approach would be to use genotypic DST and, where applicable, phenotypic DST for RIF as surrogates for RFB DST.
ᵈ Genotypic DST and, where applicable, phenotypic DST for RIF should serve as surrogates for RPT DST.
ᵉ Ethambutol 5 mg/L in MGIT is not equivalent to other methods. Ethambutol testing in 7H11 is not equivalent to 7H10. There is insufficient evidence to recommend a change in concentration for any method.
ᶠ Testing of ofloxacin is not recommended because it is no longer used to treat drug-resistant TB and laboratories should transition to testing the specific fluoroquinolones used in treatment regimens. During this transition, testing of ofloxacin at the CCs (i.e. 4.0 mg/L on LJ, 2.0 mg/L on 7H10, 2.0 mg/L on 7H11 and 2.0 mg/L in MGIT) may be performed instead of testing at the CCs for levofloxacin and moxifloxacin, but not for the CBs for moxifloxacin.
ᵍ Levofloxacin and moxifloxacin interim CCs for LJ have been established despite very limited data.
ʰ CBs for 7H10 and MGIT apply to high-dose moxifloxacin (i.e. 800 mg daily).
ᶦ Interim CCs have been established.
ʲ Cycloserine CC on LJ has been withdrawn due to limited evidence.