Line probe assays for detection of drug-resistant tuberculosis: interpretation and reporting manual for laboratory staff and clinicians

Line probe assays for detection of drug-resistant tuberculosis Interpretation and reporting manual for laboratory staff and clinicians

Line probe assays for detection of drug-resistant tuberculosis Interpretation and reporting manual for laboratory staff and clinicians Line probe assays for detection of drug-resistant tuberculosis: interpretation and reporting manual for laboratory staff and clinicians ISBN 978-92-4-004666-5 (electronic version) ISBN 978-92-4-004667-2 (print version) © World Health Organization 2022 Some rights reserved. This work is available under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons. org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non- commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. The use of the WHO logo is not permitted. 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Design by minimum graphics. iii CONTENT Contents Acknowledgements v Acronyms and abbreviations vi Glossary vii Preface viii Introduction 1 Principle of the line probe assay 2 GenoType MTBDRplus version 2 3 GenoType MTBDRs/ version 2 4 Interpretation and reporting 4 Revisions to manufacturers’ interpretations 5 Definition of additional follow-up of diagnosis to guide initiation of appropriate treatment 8 Interpretation of first-line line probe assay results 10 Rifampicin 10 Isoniazid 12 Interpretation of second-line line probe assay results 14 Fluoroquinolones 14 Amikacin 17 Assessment of drug-resistant cases based on second-line line probe assay results 19 Case 1. No resistance mutations detected or inferred in any of the genomic regions included in second-line line probe assay 19 Case 2. Detection of resistance mutations associated with high-level resistance to moxifloxacin 20 Case 3. Detection of mutations associated with at least low-level resistance to moxifloxacin 21 Case 4. Precise mutation unknown, only inferred for fluoroquinolones (i.e., gyrA and gyrB) 22 Case 5. Detection of mutations that cause resistance to amikacin 23 Case 6. Precise mutation unknown, only inferred, in the rrs region 24 Case 7. Precise mutation unknown, only inferred, in the eis region 25 Line probe assays for detection of drug-resistant tubercuLosis iv References 26 Annex 1. Reporting format for first-line line probe assay results and practical examples 28 Annex 2. Reporting format for second-line line probe assay results and practical examples 29 Annex 3. Specific nucleotide changes detected with mutation probes 31 vLIST OF ABBREvIATIONS Acknowledgements This manual is an updated version of the interpretation and reporting guide on line probe assays originally developed as a product of the Global Laboratory Initiative core group, by Elisa Tagliani (San Raffaele Scientific Institute, Milan, Italy), with contributions from Daniela Cirillo (San Raffaele Scientific Institute), Elisa Ardizzoni, Bouke de Jong and Leen Rigouts (Institute of Tropical Medicine, Antwerp, Belgium). Coordination and substantial technical input were provided by Dennis Falzon, Christopher Gilpin, Lice González-Angulo, Alexei Korobitsyn, Fuad Mirzayev and Karin Weyer of the World Health Organization (WHO) Global TB Programme during finalization of the document. We thank past and current members of the Global Laboratory Initiative core group for their extensive contribution: Olajumoke Tubi Abiola, Maka Akhalaia, Heidi Albert, Heather Alexander, Uladzimir Antonenka, Martina Casenghi, Fernanda Dockhorn, Kathleen England, Lucilaine Ferrazoli, Christopher Gilpin, Petra de Haas, Patricia Hall, Sarder Tanzir Hossain, Marguerite Massinga Loembe, Alaine Umubyeyi Nyaruhirira, Daniel Orozco, Kaiser Shen, Thomas Shinnick, Alena Skrahina, Sabira Tahseen and Hung van Nguyen. WHO appreciates the feedback provided by the following partners and stakeholders: Ignacio Monedero-Recuero of the Global Drug-resistant TB Initiative, Paolo Miotto (San Raffaele Scientific Institute), Claudio Köser (Cambridge University, United Kingdom), Natalia Shubladze (Global Laboratory Initiative core group) and Soudeh Ehsani at the WHO Regional Office for Europe. The Global Laboratory Initiative is a working group of the Stop TB Partnership. Development and publication of this document were made possible with financial support from the United States Agency for International Development. vi FIRST AND SECOND LINE DRUGS LINE PROBE ASSAYS Acronyms and abbreviations 7H10 Middlebrook 7H10 medium Am amikacin CB clinical breakpoint CC critical concentration DST drug-susceptibility testing Eto ethionamide FQ fluoroquinolone H isoniazid Lfx levofloxacin LPA line probe assay MDR-TB multidrug-resistant tuberculosis MGIT BACTEC™ Mycobacterial Growth Indicator Tube™ 960 Mfx moxifloxacin MIC minimum inhibitory concentration MTBC Mycobacterium tuberculosis complex MUT probe mutation probe Pto prothionamide PZA pyrazinamide QRDR quinolone-resistance determining region R resistant Rif rifampicin S susceptible SL-LPA second-line line probe assay TB tuberculosis WT wild type vii GLOSSARY OF TERMS Glossary Critical concentration (CC): The lowest concentration of an anti-TB agent that will inhibit the growth of 99% of phenotypically wild type isolates of Mycobacterium tuberculosis complex (MTBC) in vitro. Clinical breakpoint (CB): concentration(s) of an antimicrobial agent that defines a minimum inhibitory concentration (MIC) above the critical concentration that separates strains that are likely to respond to treatment from those that will probbably not respond. This concentration is determined by correlation with clinical outcome data, the distribution of MICs, genetic markers and data on pharmacokinetics and pharmacodynamics, including drug dose. An increased dose can be used to overcome resistance observed at lower doses, up to the maximum tolerated dose, i.e., the CB above which the drug is not recommended for use. The CB is used to guide clinical decisions in the treatment of individual patients. The CB is not applicable for surveillance of drug resistance. Minimum inhibitory concentration (MIC): The lowest concentration of an antimicrobial agent that prevents growth of more than 99% of a microorganism in a solid medium or broth dilution susceptibility test. Typically, when MICs tested in a standardized method are aggregated for one species, a single Gaussian-shaped MIC distribution is observed, which corresponds to the phenotypically wild-type (WT) distribution of that species (i.e., the distribution of organisms that lack phenotypically detectable resistance mechanisms). Additional distributions with higher overall MICs may be identified that correspond to intrinsically or naturally resistant organisms (i.e., phenotypically non-wild type distribution). viii FIRST AND SECOND LINE DRUGS LINE PROBE ASSAYS Preface This document was developed to provide practical guidance on interpretation of the most commonly used first- and second-line line probe assays (LPAs) (i.e., GenoType MTBDRplus v2.0 and GenoType MTBDRsl v2.0 assays; Bruker-Hain). In this updated manual, the interpretation of mutations identified by the two assays has been revised to align them with the most recent WHO catalogue of mutations in Mycobacterium tuberculosis complex (MTBC) and their association with drug resistance (1) and to present the latest changes in instructions for use of the assays (2). The manual is intended for both laboratory staff and clinicians. It provides information on: — the association of specific mutations detected by the most commonly used line probe assays with phenotypic drug resistance; — instances in which specific resistance-conferring mutations are not identified and resistance can only be inferred; — actions to be performed when certain mutations are detected in the assays; and — the clinical implications of specific LPA mutations for selection of appropriate tuberculosis (TB) treatment regimens. In addition, this document provides support for staff at national and regional TB reference laboratories in understanding and managing any discrepancies between phenotypic and genotypic drug susceptibility testing (DST). The manual outlines the mutations identified with both first- and second-line LPA test strips, including information on their association with phenotypic drug resistance based on the WHO catalogue of mutations in MTBC and their association with drug resistance (1) and the MICs for first- and second-line drugs reported by WHO (3, 4). Test interpretation, follow-up diagnostic testing and the clinical implications of the presence of specific mutations and inferred resistance are also described. The guide also presents case studies of examples of LPA test results and describes how results should be reported to clinicians, with recommended, customizable reporting templates (Annexes 1 and 2). 1INTRODUCTION Introduction In the past two decades, better understanding of the molecular bases of resistance to tuberculosis (TB) drugs has resulted in the development of various genotypic assays for rapid determination of resistance to anti-TB agents. Molecular testing has several advantages besides the rapidity of diagnosis, including: direct use of clinical specimens (without time-consuming solid or liquid culture to isolate Mycobacterium tuberculosis complex (MTBC) from a patient sample) and of specimens containing non- viable bacteria (e.g., bacteria killed by heat or chemical inactivation), greater potential for high-throughput testing and fewer laboratory biosafety requirements for testing procedures (i.e., low complexity-nucleic acid amplification testing). In 2008, WHO endorsed use of the first line probe assay (LPA), the GenoType MTBDRplus version 1 (referred to as GenoType MTBDRplus v1), for rapid detection of multidrug-resistant TB (MDR-TB) (5). In 2011, newer versions of the LPA technology became available, including the GenoType MTBDRplus version 2 (referred to as GenoType MTBDRplus v2) and the Nipro (Tokyo, Japan) non-tuberculous mycobacteria +MTBDR detection kit 2 (referred to as “Nipro”). The aim of these newer LPAs was to improve the sensitivity of MTBC detection and simultaneously detect resistance to rifampicin (Rif) and isoniazid (H). In 2015, the Foundation for Innovative New Diagnostics compared the Nipro and the GenoType MTBDRplus v2 LPAs with GenoType MTBDRplus v1 and found equivalence among the three commercially available LPAs for detecting MTBC and resistance to Rif and H (6). The first commercial LPA for detection of resistance to second-line TB drugs was the GenoType MTBDRsl version 1.0 (referred to as GenoType MTBDRsl v1), developed by Hain Lifescience more than a decade ago. An updated version of this assay (GenoType MTBDRsl v2) for detecting both the mutation associated with resistance to fluoroquinolone (FQ) and second-line injectable drugs detected with version 1.0 as well as other mutations (described below) became available in 2015. The following year, the WHO recommended use of the commercially available first- line LPAs (i.e., GenoType MTBDRplus v1, GenoType MTBDRplus v2 and Nipro) for initial testing instead of phenotypic drug-susceptibility testing (DST) to detect resistance to Rif and H (7). WHO also recommended use of GenoType MTBDRsl (v1 and v2) to detect resistance to FQs and amikacin (Am) in patients with Rif-resistant/MDR-TB and to guide initiation of an appropriate MDR-TB treatment regimen (8). Most recently, in 2021, the WHO recommended use of the LPA Genoscholar PZA-TB II (Nipro) for detection of resistance to pyrazinamide (PZA) in isolates from patients with bacteriologically confirmed pulmonary TB (9). For a more detailed description of the place of first- and second-line LPA within TB diagnostic algorithms, refer to module 3 of the 2021 WHO operational handbook on tuberculosis (10). This document focuses on the two currently most widely used LPAs (GenoType Line probe assays for detection of drug-resistant tubercuLosis 2 MTBDRplus v2 and GenoType MTBDRsl v2). It provides guidance to laboratory staff and clinicians in interpreting and reporting the results of both first- and second-line LPAs for better understanding and management of possible discrepancies between phenotypic and genotypic DST and the impact of LPA results on decisions about further TB testing and treatment. Principle of the line probe assay LPAs are a family of DNA strip-based tests that allow users to determine the drug resistance profile of an MTBC strain by interpreting a pattern of bands that represent lines of immobilized probes that are bound (or hybridized) to MTBC amplicons (DNA amplification products). LPA probes are designed to target the most common mutations associated with resistance to first- and second-line anti-TB agents and specific MTBC wild-type (WT) DNA sequences. LPAs are approved by WHO for rapid detection of drug resistance to first- and second-line anti-TB agents, including PZA. They can be used to test culture isolates (indirect testing, e.g., Genoscholar PZA-TB II) and for direct testing of acid-fast bacilli smear microscopy-positive specimens (first-line LPA) and both smear-positive and smear-negative sputum specimens (second-line LPA) (7, 8). Mutations are detected by binding of amplicons to probes that target the most common mutations (MUT probes) (with, e.g., first- and second-line LPAs) or by lack of amplicon binding (i.e., lack of hybridization) to the corresponding WT probes (e.g., PZA-LPA), defined as “inferred resistance”. The post-hybridization reaction leads to development of coloured bands on the strip at the site of probe binding. It is important to note that, like other molecular tests currently endorsed by WHO, LPAs have some limitations: • Although LPAs can detect the mutations most frequently identified in resistant strains, some mutations that confer resistance are outside the regions covered by the test and resistance cannot be completely excluded, even in the presence of all WT probes. Thus, in some cases additional phenotypic DST may be necessary for a full assessment of the presence of a resistant strain. • Some mutations are identified specifically by MUT probes, whereas others are inferred only by the absence of binding of the amplicons to WT probes. The lack of binding of a WT probe without simultaneous binding of an MUT probe is probably due to the presence of a resistance mutation. Systematic errors are possible if there are synonymous and non-synonymous mutations (e.g., phylogenetic mutations) (11). This is rare (< 1% of isolates), although the frequency of these isolates may increase in in certain settings (12). • LPA is less efficient than conventional culture-based DST in detecting resistance in samples that harbour both drug-susceptible and -resistant bacteria (i.e., heteroresistance). Specifically, LPA can be used to identify resistant bacteria with mutations detected by the MUT probes if resistant bacteria represent at least 5% of the total population; however, resistant bacteria with mutations inferred by the absence of WT probes would probably be missed if the resistant population represents less than 95% of the total bacterial population (13, 14). 3The overall sensitivity and specificity of LPAs for different drugs are reported in detail elsewhere (9). Briefly, first-line LPA showed a sensitivity of 95.8% and a specificity of 98.4% for detection of Rif resistance by direct testing and a sensitivity of 94.5% and a specificity of 99.3% for detection of H resistance. Second-line LPAs (GenoType MTBDRsl) had a pooled sensitivity of 86.2% and a specificity of 98.6% for the detection of FQ resistance by direct testing and a pooled sensitivity of 87.0% and a specificity of 99.5% for detection of resistance to second-line injectable drugs. PZA- LPA (Genoscholar PZA-TB II) showed a pooled sensitivity of 81.2% and a specificity of 97.8% for detection of resistance to PZA in MTBC isolates (9). Additional information on the PZA-LPA can be found in the information sheet in the annex to the 2021 WHO operational handbook on tuberculosis: Module 3 (10). GenoType MTBDRplus Version 2 GenoType MTBDRplus (Fig. 1a) targets specific mutations in the Rif resistance- determining region of the rpoB gene (from codon 505 to 533) (Fig. 2) to detect Rif resistance and mutations in the inhA promoter (from –16 to –8 nucleotides upstream) INTRODUCTION Line 1 ........ Conjugate Control 2 ........ Amplification Control 3 ........ M. tuberculosis complex TUB 4 ........ rpoB Locus Control rpoB 5 ........ rpoB wild type probe 1 rpoB WT1 6 ........ rpoB wild type probe 2 rpoB WT2 7 ........ rpoB wild type probe 3 rpoB WT3 8 ........ rpoB wild type probe 4 rpoB WT4 9 ........ rpoB wild type probe 5 rpoB WT5 10 ........ rpoB wild type probe 6 rpoB WT6 11 ........ rpoB wild type probe 7 rpoB WT7 12 ........ rpoB wild type probe 8 rpoB WT8 13 ........ rpoB mutation probe 1 rpoB MUT1 14 ........ rpoB mutation probe 2A rpoB MUT2A 15 ........ rpoB mutation probe 2B rpoB MUT2B 16 ........ rpoB mutation probe 3 rpoB MUT3 17 ........ katG Locus Control katG 18 ........ katG wild type probe katG WT 19 ........ katG mutation probe 1 katG MUT1 20 ........ katG mutation probe 2 katG MUT2 21 ........ inhA Locus Control inhA 22 ........ inhA wild type probe 1 inhA WT1 23 ........ inhA wild type probe 2 inhA WT2 24 ........ inhA mutation probe 1 inhA MUT1 25 ........ inhA mutation probe 2 inhA MUT2 26 ........ inhA mutation probe 3A inhA MUT3A 27 ........ inhA mutation probe 3B inhA MUT3B Colored marker Line 1 ........ Conjugate Control 2 ........ Amplification Control 3 ........ M. tuberculosis complex TUB 4 ........ gyrA Locus Control gyrA 5 ........ gyrA wild type probe 1 gyrA WT1 6 ........ gyrA wild type probe 2 gyrA WT2 7 ........ gyrA wild type probe 3 gyrA WT3 8 ........ gyrA mutation probe 1 gyrA MUT1 9 ........ gyrA mutation probe 2 gyrA MUT2 10 ........ gyrA mutation probe 3A gyrA MUT3A 11 ........ gyrA mutation probe 3B gyrA MUT3B 12 ........ gyrA mutation probe 3C gyrA MUT3C 13 ........ gyrA mutation probe 3D gyrA MUT3D 14 ........ gyrB Locus Control gyrB 15 ........ gyrB wild type probe gyrB WT 16 ........ gyrB mutation probe 1 gyrB MUT1 17 ........ gyrB mutation probe 2 gyrB MUT2 18 ........ rrs Locus Control rrs 19 ........ rrs wild type probe 1 rrs WT1 20 ........ rrs wild type probe 2 rrs WT2 21 ........ rrs mutation probe 1 rrs MUT1 22 ........ rrs mutation probe 2 rrs MUT2 23 ........ eis Locus Control eis 24 ........ eis wild type probe 1 eis WT1 25 ........ eis wild type probe 2 eis WT2 26 ........ eis wild type probe 3 eis WT3 27 ........ eis mutation probe 1 eis MUT1 Colored marker Fig. 1. Configuration of GenoType MTBDRplus V2 a and GenoType MTBDRsl V2 b strips a (2) b (15) Line probe assays for detection of drug-resistant tubercuLosis 4 and katG (codon 315) genomic regions to identify resistance to H (2). The specific nucleotide changes detected by the test are reported in Annex 3. GenoType MTBDRsl Version 2 The second version of GenoType MTBDRsl (Fig. 1b) includes the quinolone-resistance determining region (QRDR) of gyrA (from codon 85 to 96) (Fig. 3) and of gyrB (from codon 536 to 541) genes for detection of resistance to FQs and the rrs (nucleic acid positions 1401, 1402 and 1484) and the eis promoter regions (from –37 to –2 nucleotides upstream) for detection of resistance to Am (15). The precise regions covered by all MUT probes have not been disclosed, and only some of the regions covered by WT probes are known (see Fig. 2 and 3). The specific nucleotide changes detected by the MUT probes are reported in Annex 3. Interpretation and reporting The LPA has two internal controls on the strip: “conjugate control” (line 1) and “amplification control” (line 2) (Fig. 1). The conjugate control line should always be visible in order to document the efficiency of conjugate binding and substrate reaction. The amplification control line serves as reference for interpretation of WT and MUT probes: only those bands of which the intensity is about a strong as or stronger than that of the amplification control line should be considered. In the case of a positive test result (i.e., a positive M. tuberculosis control band), the signal of the amplification control zone may be weak or even vanish. This may occur more frequently in indirect testing. The absence of amplification control may be due to competition among single reactions during amplification. This indicates that the test has been conducted correctly and need not be repeated with the same sample. In first-line LPA, when there are strong signals of WT bands but weak or no staining of the amplification control band, a single WT band that is significantly fainter than the remaining WT bands of the same locus (or the Locus control for katG) should be considered negative (2). In the case of a negative test result, both the conjugate and the amplification control bands should always be visible to ensure a valid negative result. The absence of an amplification control band in a negative test indicates mistakes during setup and/or performance of the amplification reaction or the presence of amplification inhibitors. In this case, the test result is invalid and must be repeated. The M. tuberculosis control band (line 3) is present only if the DNA amplified is from members of the MTBC. In rare cases, the M. tuberculosis control band band is missing because of competition among the single amplification reactions during the polymerase chain reaction. When an evaluable resistance pattern develops, however, the presence of an MTBC strain should be suspected, and the test should be repeated. In rare cases in direct testing, only the conjugate and amplification control bands and the M. tuberculosis control band may be visible, in the absence of an evaluable resistance pattern. This may indicate the presence of an MTBC strain at very low concentration, below the limit of detection. In such instances, the test should be repeated on the corresponding culture isolate (i.e., indirect testing). The presence of non-tuberculous mycobacteria in the specimen can result in random banding patterns, with several species testing positive at some rpoB WT bands because of gene similarity among the species. In the presence of non-tuberculous rather that 5MTBC bacteria, the M. tuberculosis control band will always be absent, and the result should be reported as “MTBC not detected”. The gene locus control bands for the different target regions analysed on the DNA strip are located just before their respective WT and MUT probe bands. The locus control bands must always be present in order for the assay to be considered valid for the corresponding target. In rare cases, all bands of a gene locus (including the locus control band) may be missing. In direct testing, such a banding pattern cannot be evaluated, and the test must be repeated. In indirect testing, however, the complete absence of the katG locus indicates resistance to H of the strain tested due to mutations or deletions in the locus control region or to complete or partial deletion of the gene (2). The WT reaction zones comprise regions of the genome with known resistance mutations. The MUT probe reaction zones correspond to probes that identify the most common resistance mutations of the gene being examined. Resistance is detected when MUT probes are developed, whereas, in the absence of WT probes, resistance can only be inferred (see below for details). Concomitant detection of all WT probes and any of the MUT probes in the corresponding target region indicates the presence of heteroresistance (i.e., susceptible and resistant bacteria in the same sample). In this case, the result should be reported as “resistant”. Revisions to manufacturers’ interpretations (2, 15) Use of the term “Resistance not detected” instead of “Susceptible” to define the bacterial resistance profile Given the limitations of LPA and in particular the fact that resistance cannot be totally excluded even in the presence of all WT probes (as not all mutations that confer resistance are covered by these tests, and mutations that are covered may occur below the limit of detection), it is most appropriate to report the result as “Resistance detected” or “Resistance not detected”. Differentiation of resistance into “Resistance inferred” and “Resistance detected” The term “Resistance inferred” is used when one or more WT probes in regions of the gene known to confer resistance to the drug are not developed, and none of the MUT probes in the corresponding region is developed. In this case, only the region in which the mutation is located and not the precise mutation can be reported. The term “Resistance detected” is used when one or more MUT probes that identify specific mutations conferring resistance to the drugs are developed (regardless of whether WT probes are developed). Stratification of resistance mutations for H and moxifloxacin (Mfx) into mutations associated with “low-level resistance” and “high-level resistance” Mutations that confer resistance to H and Mfx are stratified into those associated with low- and high-level resistance and, depending on the distribution of the associated MICs, with a low and a high increase in MIC, respectively. This stratification has important implications for the inclusion of H and Mfx in a treatment regimen, as resistance due to mutations associated with low-level resistance for H or Mfx may be overcome by increasing the drug dose. For H, in-vitro evidence suggests that when specific inhA promoter mutations, INTRODUCTION Line probe assays for detection of drug-resistant tubercuLosis 6 which are generally associated with low-level resistance, are detected (in the absence of any katG mutation), increasing the drug dose might be effective; thus, administration of H at a maximum dose of 15 mg/kg per day could be considered. In the case of katG mutations, which are more commonly associated with high-level resistance, administration of H at an even higher dose is less likely to be effective. The presence of combined mutations in the inhA promoter and the katG gene results in substantial increases in the MIC (i.e., high-level resistance), which is unlikely to be compensated for by increasing the dose (17). For Mfx, if mutations associated with MIC increase above the CC but below the CB, which are defined as mutations associated with low-level resistance, high-dose Mfx (up to 800 mg daily for adults) might be effective. When resistance to Mfx is inferred (i.e., the specific mutation is unknown), the presence of mutations associated with at least low-level resistance is inferred, and therefore a high dose of Mfx might still be effective. In this case, however, it is recommended that DST be performed for Mfx at the CB, and, if available, sequencing be conducted to determine the specific mutation. If the MTBC strain is resistant to Mfx at the CB because of the presence of mutations associated with high-level resistance, the drug cannot be considered effective. When more than one probe per drug provides information (e.g., concomitant detection of mutations associated with different resistance levels), the criterion for interpretation is that the mutations associated with high-level resistance overrule mutations associated with low-level resistance. Similarly, mutations detected by MUT probes overrule mutations that are only inferred by the absence of WT probes. In summary, results should be reported according to the following hierarchy (where the “>” sign means “overrule”): • For H: Mutation associated with high-level resistance detected > Mutation associated with high-level resistance inferred > Mutation associated with at least low-level resistance detected > Mutation associated with at least low-level resistance inferred > Resistance not detected • For Mfx: Mutation associated with high-level resistance detected > Mutation associated with at least low-level resistance detected > Mutation associated with at least low-level resistance inferred > Resistance not detected • For Rif, levofloxacin (Lfx), Am, kanamycin and capreomycin: Resistance detected > Resistance inferred > Resistance not detected In summary, according to whether WT and MUT probes are present, the following four cases may occur: 7Case WT reaction zones MUT probe reaction zones Interpretation 1 All WT probes are developed. All MUT probes are not developed. Resistance not detected 2 One or more WT probes are not developed. One or more MUT probes in the corresponding region are developed. Depending on the drug: • Resistance detected (Rif, Am) • Mutations associated with high-level resistance detected (H and Mfx) • Mutations associated with at least low- level resistance detected (H and Mfx) 3 One or more WT probes are not developed. No MUT probes are developed. Depending on the drug: • Resistance inferred (Rif, Am) • Mutations associated with high-level resistance inferred (H and Mfx) • Mutations associated with at least low- level resistance inferred (H and Mfx) 4 All WT probes are developed. One MUT probe is developed. Resistance detected (due to heteroresistance); interpret according to case 2. Interpretation of resistance profile for amikacin The WHO Catalogue of mutations in MTBC (1) defines two additional markers for Am resistance: (i) the eis c-14t mutation, identified by the eis MUT1 probe, is classified as a definitive marker for resistance (group 1); and (ii) the rrs c1402t, inferred by the absence of the rrs WT1, is recognized as a group 2 mutation (i.e., a mutation associated with resistance-interim). Therefore, the interpretation of second-line-LPA for Am has been revised accordingly. Exclusion of the eis WT3 probe To date, there is no clear evidence that the mutation c-2a in the eis promoter region is on its own a valid marker of resistance (16). Therefore, if the eis WT3 probe is not developed, the test interpretation for kanamycin has been revised to “Resistance not detected”. Interpretation of resistance profiles for ethionamide and prothionamide Mutations leading to an overexpression of inhA gene, such those detected by first- line LPA, are associated with resistance to ethionamide (Eto) (1) and prothionamide (Pto). Therefore, if these mutations are detected, resistance to the two drugs should be reported, and they should be excluded from the treatment regimen. Even in the absence of mutations in the inhA promoter region, however, resistance to Eto and Pto cannot be excluded. Mutations conferring resistance to these drugs may in fact be present in genomic regions not targeted by LPA (e.g., ethA, ethR) (1). Reporting of results for kanamycin and capreomycin WHO currently recommends that injectable medicines be phased out as a priority in all treatment regimens and be replaced by bedaquiline, which makes rapid DST for second-line injectables unnecessary (17). In addition, since 2018, WHO no longer recommends use of kanamycin or capreomycin because of the increased risks of INTRODUCTION Line probe assays for detection of drug-resistant tubercuLosis 8 treatment failure and relapse associated with their use in longer MDR-TB regimens (18). Am is the only second-line injectable agent still recommended for use in MDR- TB regimens when options for composition of the treatment regimen are limited (17). Therefore, interpretation of second-line LPA for kanamycin and capreomycin is not considered in this manual. Definition of additional follow-up diagnosis to guide initiation of appropriate treatment Depending on the region interrogated by first- and second-line LPA, one or more follow-up diagnostic actions are either recommended or suggested to guide treatment regimens. A decision to conduct the optional follow-up diagnostic actions should be guided by considerations of the risk group of the patient for resistance and by the prevalence of resistance in the setting, as these factors affect the positive predictive value of the test. The follow-up diagnostic actions that are recommended or suggested depend on the drug. The actions are summarized briefly below. Rifampicin (Rif): • If resistance is inferred from the absence of binding of the amplicons to WT probes (i.e., one or more WT probes not developed), sequencing of the rpoB gene is suggested to identify the specific mutation. For interpretation of rpoB mutations, see the WHO catalogue (1). Isoniazid (H): • If resistance is inferred from the absence of binding of the amplicons to WT probes in the katG region (i.e., one or more WT probes not developed), sequencing of the katG gene is suggested to identify the specific mutation. For interpretation of katG mutations, see the WHO catalogue (1). • If mutations associated with low-level resistance are detected (i.e., MUT probes developed in the inhA promoter region in the absence of mutations in the katG target region), sequencing of the inhA coding region and the katG gene is suggested, because the concomitant presence of additional mutations in the inhA coding region or in positions other than 315 in the katG gene (mutations not detected by GenoType MTBDRplus) (19, 20), which are globally rare but could be more frequent in some settings, may substantially increase the MIC to a level too high to be compensated for by increasing the dose of the drug. • If mutations associated with low-level resistance are inferred from the absence of binding of the amplicons to WT probes in the inhA promoter region (and no mutations are detected in the katG target region), it is recommended that the test be repeated to confirm the result. Optional follow-up diagnostic actions include sequencing of the inhA promoter to identify the specific mutation or performing phenotypic DST for H. 9Moxifloxacin (Mfx): • If mutations associated with low-level resistance are detected (i.e., MUT1, MUT2, MUT3A probes developed in gyrA and/or MUT1, MUT2 probes developed in gyrB regions), phenotypic DST for Mfx is recommended to exclude resistance at CB. • If mutations associated with low-level resistance are inferred from the absence of binding of the amplicons to WT probes in the gyrA or gyrB region (i.e., WT probes not developed), phenotypic DST for Mfx is recommended to exclude resistance at CB. Optional follow-up actions include sequencing of gyrA and/or gyrB QRDR to identify the specific mutation and/or phenotypic DST for Mfx (and/or Lfx) at CC (depending on laboratory capacity). Amikacin (Am): • If resistance is inferred from the absence of binding of the amplicons to WT probes in the rrs region (i.e., one or more WT probes are not developed), it is recommended that testing be repeated to confirm the result. Sequencing of rrs gene is suggested to identify the specific mutation. • If resistance is inferred from the absence of binding of the amplicons to the WT2 probe in the eis region and no MUT1 probe is developed, it is recommended that testing be repeated to confirm the result. Sequencing of the eis gene, including the promoter region, is suggested to identify the specific mutation. INTRODUCTION Line probe assays for detection of drug-resistant tubercuLosis 10 In te rp re ta tio n of fi rs t- lin e lin e pr ob e as sa y re su lts Ri fa m pi ci n Th e Ri f r es is ta nc e- de te rm in in g re gi on o f t he rp oB g en e, c od on s co ve re d by th e W T pr ob es a nd th e sp ec ifi c m ut at io ns re co gn iz ed b y th e M U T pr ob es in M TB D R pl us v er 2 .0 (2 )- fo r E . c ol i v s. M TB c od on n um be rin g an d am in o ac id n om en cl at ur e ar e sh ow n in F ig . 2 . O ve ra ll, th e sp ec ifi ci ty of M TB D R pl us v er 2 .0 fo r Ri f r es is ta nc e is v er y go od . I f t he v al id ity o f a R if re si st an ce r es ul t is d ou bt fu l, re qu es t rp oB s eq ue nc in g as t he g ol d st an da rd . Fi g. 2 . R if am p ic in r es is ta n ce -d et er m in in g re gi o n in te rr o ga te d b y G en oT yp e M TB D R p lu s Ta rg e t re g io n M TB D R p lu s p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic a ct io n a C lin ic al im p lic at io n s rp oB W T1 rp oB W T1 n ot d ev el op ed M ut at io n (s ) i n co do ns 5 05 –5 09 (4 24 –4 28 )b Re si st an ce to rif am pi ci n (R if) in fe rr ed O p ti o n al : P er fo rm s eq ue nc in g of r po B to id en tif y th e sp ec ifi c m ut at io n. Ri f i s no t e ff ec tiv ec rp oB W T2 rp oB W T2 n ot d ev el op ed M ut at io n (s ) i n co do ns 5 10 -5 13 (4 29 –4 32 )b Re si st an ce to R if in fe rr ed O p ti o n al : P er fo rm s eq ue nc in g of r po B to id en tif y th e sp ec ifi c m ut at io n. Ri f i s no t e ff ec tiv ec rp oB W T2 /3 rp oB W T2 a nd W T3 n o t de ve lo pe d M ut at io n (s ) i n co do ns 5 10 –5 17 (4 29 –4 36 )b Re si st an ce to R if in fe rr ed O p ti o n al : P er fo rm s eq ue nc in g of r po B to id en tif y th e sp ec ifi c m ut at io n. Ri f i s no t e ff ec tiv ec rp oB W T3 /4 rp oB M U T1 d ev el op ed D 51 6v (D 43 5v )b Re si st an ce to R if de te ct ed N o ad di tio na l d ia gn os tic a ct io n re qu ire d. Ri f i s no t e ff ec tiv e 50 5 50 8 50 9 51 1 51 3 51 4 51 5 51 6 51 8 52 2 52 6 53 1 53 3 M U T 1 D 51 6V ( D 4 35 V ) M U T 2 A H 52 6Y ( H 4 45 Y ) M U T 2B H 52 6D ( H 4 45 D ) M U T 3 S 53 1L ( S 45 0 L) rp oB W T1 rp oB W T3 rp oB W T5 rp oB W T7 rp oB W T8 rp oB W T6 rp oB W T4 rp oB W T2 11 Ta rg e t re g io n M TB D R p lu s p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic a ct io n a C lin ic al im p lic at io n s rp oB W T3 , W T4 a nd M U T1 no t d ev el op ed M ut at io n (s ) i n co do ns 5 13 –5 19 (4 32 –4 38 )b Re si st an ce to R if in fe rr ed O p ti o n al : P er fo rm s eq ue nc in g of r po B to id en tif y th e sp ec ifi c m ut at io n. Ri f i s no t e ff ec tiv ec rp oB W T4 /5 rp oB W T4 a nd W T5 n o t de ve lo pe d M ut at io n (s ) i n co do ns 5 16 –5 22 (4 35 –4 41 )b Re si st an ce to R if in fe rr ed O p ti o n al : P er fo rm s eq ue nc in g of r po B to id en tif y th e sp ec ifi c m ut at io n. Ri f i s no t e ff ec tiv ec . rp oB W T5 /6 rp oB W T5 a nd W T6 n ot de ve lo pe d M ut at io n (s ) i n co do ns 5 18 –5 25 (4 37 –4 44 )b Re si st an ce to R if in fe rr ed O p ti o n al :P er fo rm s eq ue nc in g of r po B to id en tif y th e sp ec ifi c m ut at io n. Ri f i s no t e ff ec tiv ec rp oB W T7 rp oB M U T2 A d ev el op ed H 52 6Y (H 44 5Y )b Re si st an ce to R if de te ct ed N o ad di tio na l d ia gn os tic a ct io n re qu ire d Ri f i s no t e ff ec tiv e rp oB M U T2 B d ev el op ed H 52 6D (H 44 5D )b Re si st an ce to R if de te ct ed N o ad di tio na l d ia gn os tic a ct io n re qu ire d Ri f i s no t e ff ec tiv e rp oB W T7 , M U T2 A a nd M U T2 B n ot d ev el op ed M ut at io n (s ) i n co do ns 5 26 –5 29 (4 45 –4 48 )b Re si st an ce to R if in fe rr ed O p ti o n al : P er fo rm s eq ue nc in g of r po B to id en tif y th e sp ec ifi c m ut at io n. Ri f i s no t e ff ec tiv ec rp oB W T8 rp oB M U T3 d ev el op ed S5 31 L (S 45 0L )b Re si st an ce to R if de te ct ed N o ad di tio na l d ia gn os tic a ct io n re qu ire d Ri f i s no t e ff ec tiv e rp oB W T8 a nd M U T3 n o t de ve lo pe d M ut at io n (s ) i n co do ns 5 30 –5 33 (4 49 –4 52 )b Re si st an ce to R if in fe rr ed O p ti o n al : P er fo rm s eq ue nc in g of r po B to id en tif y th e sp ec ifi c m ut at io n. Ri f i s no t e ff ec tiv ec a Th e de ci si on t o pe rf or m t he o pt io na l d ia gn os tic a ct io ns s ho ul d be g ui de d by c on si de ra tio ns o f t he p at ie nt ’s r is k gr ou p fo r re si st an ce a nd b y th e pr ev al en ce o f r es is ta nc e in t he s et tin g, a s th es e fa ct or s af fe ct t he p os iti ve p re di ct iv e va lu e of t he t es t. S ile nt m ut at io ns m ay b e of g re at er c on ce rn in lo w -r es is ta nt s et tin gs . b M TB c od on n um be ri ng a cc or di ng t o A nd re e t a l. (2 1) is r ep or te d in p ar en th es es . c Th is r ec om m en da tio n do es n ot a pp ly if s eq ue nc in g id en tifi es a s ile nt m ut at io n. INTERPRETATION OF FIRST-LINE LINE PROBE ASSAY RESULTS Line probe assays for detection of drug-resistant tubercuLosis 12 Is on ia zi d Ta rg e t re g io n M TB D R p lu s p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic a ct io n a C lin ic al im p lic at io n s ka tG W T ka tG M U T1 o r M U T2 de ve lo pe d S3 15 T1 / S3 15 T2 M ut at io n as so ci at ed w ith h ig h- le ve l r es is ta nc e de te ct ed . N o ad di tio na l d ia gn os tic a ct io n re qu ire d H is u nl ik el y to b e ef fe ct iv e ev en at a h ig h do se (1 7) . ka tG W T, M U T1 an d M U T2 n o t de ve lo pe db M ut at io n (s )in c od on 31 5 re gi on M ut at io n as so ci at ed w ith h ig h- le ve l r es is ta nc e in fe rr ed . O p ti o n al : P er fo rm s eq ue nc in g of k at G to id en tif y th e sp ec ifi c m ut at io n. H is u nl ik el y to b e ef fe ct iv e ev en at a h ig h do se (1 7) . in hA W T1 in hA M U T1 de ve lo pe d c- 15 t M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l r es is ta nc e de te ct ed . Re si st an ce to E to a nd P to de te ct ed . O p ti o n al :c P er fo rm s eq ue nc in g of in hA c od in g re gi on a nd k at G ge ne . N o ad di tio na l d ia gn os tic a ct io n fo r Et o an d P to H a t h ig h do se is li ke ly to b e ef fe ct iv e (1 7) . Et o an d P to a re n ot e ff ec tiv e. in hA M U T2 de ve lo pe d a- 16 gd M ut at io n lik el y as so ci at ed w ith at le as t l ow -le ve l r es is ta nc e de te ct ed . Re si st an ce to E to a nd P to de te ct ed . O p ti o n al :c P er fo rm s eq ue nc in g of in hA c od in g re gi on a nd k at G ge ne . N o ad di tio na l d ia gn os tic a ct io n fo r Et o an d P to H a t a h ig h do se is li ke ly to b e ef fe ct iv e. Et o an d P to a re n ot e ff ec tiv e. In hA W T1 , M U T1 a nd M U T2 n ot d ev el op ed M ut at io n (s ) i n th e –1 5 re gi on d M ut at io n is li ke ly to b e as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce (i nf er re d ). Re si st an ce to E to a nd P to in fe rr ed R ec o m m en d e d : R ep ea t se co nd -li ne -L PA to c on fir m th e re su lt. O p ti o n al : P er fo rm s eq ue nc in g to id en tif y sp ec ifi c m ut at io n. H a t h ig h do se is li ke ly to b e ef fe ct iv e (1 7) . Et o an d P to a re u nl ik el y to b e ef fe ct iv e. in hA W T2 In hA M U T3 A de ve lo pe d t- 8c d M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l r es is ta nc e de te ct ed . Re si st an ce to E to a nd P to de te ct ed . O p ti o n al :c P er fo rm s eq ue nc in g of in hA c od in g re gi on a nd k at G ge ne . N o ad di tio na l d ia gn os tic a ct io n fo r Et o an d P to H a t a h ig h do se is li ke ly to b e ef fe ct iv e (1 7) . Et o an d P to a re n ot e ff ec tiv e. 13 Ta rg e t re g io n M TB D R p lu s p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic a ct io n a C lin ic al im p lic at io n s In hA M U T3 B de ve lo pe d t- 8a d M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l r es is ta nc e de te ct ed Re si st an ce to E to a nd P to de te ct ed O p ti o n al :c P er fo rm s eq ue nc in g of in hA c od in g re gi on a nd k at G ge ne . N o ad di tio na l d ia gn os tic a ct io n fo r Et o an d P to H a t a h ig h do se is li ke ly to b e ef fe ct iv e (1 7) . Et o an d P to a re n ot e ff ec tiv e. In hA W T2 , M U T3 A an d M U T3 B n o t de ve lo pe d M ut at io n (s ) i n th e –8 r eg io nd M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l r es is ta nc e in fe rr ed Re si st an ce to E to a nd P to in fe rr ed R ec o m m en d e d : R ep ea t fi rs t- lin e LP A to c on fir m th e re su lt. O p ti o n al : P er fo rm s eq ue nc in g to id en tif y sp ec ifi c m ut at io n. H a t a h ig h do se is li ke ly to b e ef fe ct iv e (1 7) . Et o an d P to a re u nl ik el y to b e ef fe ct iv e. a Th e de ci si on t o pe rf or m t he o pt io na l f ol lo w -u p di ag no st ic a ct io ns s ho ul d be g ui de d by c on si de ra tio n of t he in di vi du al p at ie nt ’s r is k gr ou p fo r re si st an ce a nd b y th e pr ev al en ce o f r es is ta nc e in t he s et tin g, as t he se fa ct or s af fe ct t he p os iti ve p re di ct iv e va lu e of t he t es t. b Pa rt ia l o r w ho le d el et io n of t he k at G g en e, w hi ch is a ss oc ia te d w ith h ig h- le ve l r es is ta nc e, r es ul ts in c om pl et e ab se nc e of k at G lo cu s ba nd s (i. e. , k at G lo cu s co nt ro l, W T an d M U T pr ob es a re n ot d ev el op ed ). c Th e co nc om ita nt p re se nc e of a dd iti on al m ut at io ns in t he in hA c od in g re gi on o r in p os iti on s ot he r th an 3 15 in t he k at G g en e (m ut at io ns n ot d et ec te d by G en oT yp e M TB D R pl us ) ( 19 , 2 0) , w hi ch a re g lo ba lly ra re b ut c ou ld b e m or e fr eq ue nt in s om e se tt in gs , m ay c au se s ub st an tia l i nc re as es in t he M IC , t oo h ig h to b e co m pe ns at ed fo r by in cr ea se d th e do se o f t he d ru g. d A dd iti on al d at a co rr el at in g th es e m ut at io ns w ith p he no ty pi c D ST fo r is on ia zi d is n ee de d to in cr ea se t he c on fid en ce in t he a ss oc ia tio n of t he se m ut at io ns w ith d ru g re si st an ce . INTERPRETATION OF FIRST-LINE LINE PROBE ASSAY RESULTS Line probe assays for detection of drug-resistant tubercuLosis 14 In te rp re ta tio n of s ec on d- lin e lin e pr ob e as sa y re su lts Fl uo ro qu in ol on es Th e Q RD R of t he g yr A g en e, t he c od on s co ve re d by t he W T pr ob es a nd t he s pe ci fic m ut at io ns ( bo th a m in o ac id a nd n uc le ot id e ch an ge s) re co gn iz ed b y th e M U T pr ob es in G en oT yp e M TB D Rs l v er 2 .0 a re s ho w n in F ig . 3 . Fi g. 3 . Q u in o lo n e re si st an ce -d et er m in in g re gi o n o f gy rA g en e in te rr o ga te d w it h G en oT yp e M TB D R sl Ta rg e t re g io n M TB D R sl p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic a ct io n a C lin ic al im p lic at io n s gy rA W T 1 gy rA W T1 n o t de ve lo pe d M ut at io n (s ) i n co do ns 8 5– 89 Re si st an ce to L fx in fe rr ed M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce fo r M fx in fe rr ed R ec o m m en d e d : P er fo rm ph en ot yp ic D ST fo r M fx a t C B to e xc lu de r es is ta nc e. O p ti o n al : • Pe rf or m s eq ue nc in g of g yr A Q RD R to id en tif y sp ec ifi c m ut at io n. • Pe rf or m p he no ty pi c D ST fo r Lf x an d M fx a t C C . Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . N o te . T he se r ec om m en da tio ns d o no t a pp ly if se qu en ci ng , a va ila bl e be fo re tr ea tm en t in iti at io n, id en tifi es m ut at io ns n ot a ss oc ia te d w ith r es is ta nc e to F Q o r if ph en ot yp ic D ST sh ow s su sc ep tib ili ty a t C C . 85 86 87 88 89 90 91 92 93 94 95 96 97 M U T1 A 9 0V g cg > g tg M U T 3A D 94 A g ac > gc c M U T 3B D 94 N g ac > aa c M U T 3B D 94 Y g ac > ta c M U T 3C D 94 G g ac > g gc M U T 3D D 94 H g ac > ca c gy rA W T1 gy rA W T3 gy rA W T2 M U T2 S 91 P t cg > cc g 15 INTERPRETATION OF SECOND-LINE LINE PROBE ASSAY RESULTS Ta rg e t re g io n M TB D R sl p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic a ct io n a C lin ic al im p lic at io n s gy rA W T2 gy rA M U T1 de ve lo pe d A 90 v Re si st an ce to L fx d et ec te d. M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce to M fx de te ct ed . R ec o m m en d e d : P er fo rm ph en ot yp ic D ST fo r M fx a t C B to e xc lu de r es is ta nc e. Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . gy rA M U T2 de ve lo pe d S9 1P Re si st an ce to L fx d et ec te d. M ut at io n as so ci at ed w ith at le as t l ow -r es is ta nc e fo r M fx d et ec te d. R ec o m m en d e d : P er fo rm ph en ot yp ic D ST fo r M fx a t C B to e xc lu de r es is ta nc e. Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . gy rA W T2 , M U T1 an d M U T2 n o t de ve lo pe d M ut at io n (s ) i n co do ns 8 9– 93 Re si st an ce to L fx in fe rr ed . M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce fo r M fx in fe rr ed . R ec o m m en d e d : P er fo rm ph en ot yp ic D ST fo r M fx a t C B to e xc lu de r es is ta nc e. O p ti o n al : • Pe rf or m s eq ue nc in g of g yr A Q RD R to id en tif y sp ec ifi c m ut at io n • Pe rf or m p he no ty pi c D ST fo r Lf x an d M fx a t C C . Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . N o te . T he se r ec om m en da tio ns d o no t a pp ly if se qu en ci ng , a va ila bl e be fo re tr ea tm en t in iti at io n, id en tifi es m ut at io ns n ot a ss oc ia te d w ith r es is ta nc e to F Q o r if ph en ot yp ic D ST sh ow s su sc ep tib ili ty a t C C . gy rA W T3 gy rA M U T3 A de ve lo pe d D 94 A Re si st an ce to L fx d et ec te d. M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce fo r M fx de te ct ed . R ec o m m en d e d : P er fo rm ph en ot yp ic D ST fo r M fx a t C B to e xc lu de r es is ta nc e. Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . gy rA M U T3 B de ve lo pe d D 94 N o r D 94 Y Re si st an ce to L fx d et ec te d. M ut at io n as so ci at ed w ith hi gh -le ve l r es is ta nc e fo r M fx d et ec te d. N o ad di tio na l d ia gn os tic a ct io n re qu ire d. Le fx is n ot e ff ec tiv e. M fx is n ot e ff ec tiv e. Line probe assays for detection of drug-resistant tubercuLosis 16 Ta rg e t re g io n M TB D R sl p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic a ct io n a C lin ic al im p lic at io n s gy rA W T3 gy rA M U T3 C de ve lo pe d D 94 G Re si st an ce to L fx d et ec te d. M ut at io n as so ci at ed w ith hi gh -le ve l r es is ta nc e fo r M fx d et ec te d. N o ad di tio na l d ia gn os tic a ct io n re qu ire d. Lf x is n ot e ff ec tiv e. M fx is n ot e ff ec tiv e. gy rA M U T3 D de ve lo pe d D 94 H Re si st an ce to L fx d et ec te d. M ut at io n as so ci at ed w ith hi gh -le ve l r es is ta nc e fo r M fx d et ec te d. N o ad di tio na l d ia gn os tic a ct io n re qu ire d. Lf x is n ot e ff ec tiv e. M fx is n ot e ff ec tiv e. gy rA W T3 , M U T3 A , M U T3 B, M U T3 C a nd M U T3 D n o t de ve lo pe d M ut at io n (s ) i n co do ns 9 2– 96 Re si st an ce to L fx in fe rr ed . M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce fo r M fx in fe rr ed . R ec o m m en d e d : P er fo rm ph en ot yp ic D ST fo r M fx a t C B to e xc lu de r es is ta nc e. O p ti o n al : • Pe rf or m s eq ue nc in g of g yr A Q RD R to id en tif y sp ec ifi c m ut at io n • Pe rf or m p he no ty pi c D ST fo r Lf x an d M fx a t C C . Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . N o te . T he se r ec om m en da tio ns d o no t a pp ly if se qu en ci ng , a va ila bl e be fo re tr ea tm en t in iti at io n, id en tifi es m ut at io ns n ot a ss oc ia te d w ith r es is ta nc e to F Q , o r if ph en ot yp ic D ST sh ow s su sc ep tib ili ty a t C C . gy rB W T gy rB M U T1 de ve lo pe d N 53 8D (N 49 9D )b Re si st an ce to L fx d et ec te d. M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce fo r M fx de te ct ed . R ec o m m en d e d :P er fo rm ph en ot yp ic D ST fo r M fx to ex cl ud e re si st an ce a t C B . Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . gy rB M U T2 de ve lo pe d E5 40 v (E 50 1v )b Re si st an ce to L fx d et ec te d. M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce fo r M fx de te ct ed . R ec o m m en d e d : P er fo rm ph en ot yp ic D ST fo r M fx to ex cl ud e re si st an ce a t C B . Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . 17 Ta rg e t re g io n M TB D R sl p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic a ct io n a C lin ic al im p lic at io n s gy rB W T, M U T1 an d M U T2 n o t de ve lo pe d M ut at io n (s ) i n co do ns 5 36 –5 41 (4 97 –5 02 )b Re si st an ce to L fx in fe rr ed . M ut at io n as so ci at ed w ith a t l ea st lo w -le ve l re si st an ce fo r M fx in fe rr ed . R ec o m m en d e d : P er fo rm ph en ot yp ic D ST fo r M fx to ex cl ud e re si st an ce a t C B . O p ti o n al : • Pe rf or m s eq ue nc in g of g yr A Q RD R to id en tif y sp ec ifi c m ut at io n; • Pe rf or m p he no ty pi c D ST fo r Lf x an d M fx a t C C . Lf x is n ot e ff ec tiv e. M fx c ou ld b e us ed a t a h ig he r do se . T he re gi m en s ho ul d be r e- ev al ua te d ac co rd in g to ph en ot yp ic D ST r es ul ts a t C B . N o te . T he se r ec om m en da tio ns d o no t a pp ly if se qu en ci ng , a va ila bl e be fo re tr ea tm en t in iti at io n, id en tifi es m ut at io ns n ot a ss oc ia te d w ith r es is ta nc e to F Q , o r if ph en ot yp ic D ST sh ow s su sc ep tib ili ty a t C C . a Th e de ci si on t o pe rf or m t he o pt io na l f ol lo w -u p di ag no st ic a ct io ns s ho ul d be g ui de d by c on si de ra tio n of t he in di vi du al p at ie nt ’s r is k gr ou p fo r re si st an ce a nd b y th e pr ev al en ce o f r es is ta nc e in t he s et tin g, as t he se fa ct or s af fe ct t he p os iti ve p re di ct iv e va lu e of t he t es t. b C od on n um be ri ng s ys te m a cc or di ng t o C am us e t a l. (2 2) in p ar en th es es A m ik ac in a Ta rg e t re g io n M TB D R sl p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic ac ti o n b C lin ic al im p lic at io n s rr s W T1 rr s M U T1 d ev el op ed a1 40 1g Re si st an ce to A m d et ec te d N o ad di tio na l d ia gn os tic ac tio n re qu ire d A m is n ot e ff ec tiv e. rr s W T1 a nd M U T1 n ot de ve lo pe d M ut at io n (s ) i n th e 14 00 re gi on Re si st an ce to A m in fe rr ed O p ti o n al : P er fo rm se qu en ci ng to id en tif y sp ec ifi c m ut at io n. A m is u nl ik el y to b e ef fe ct iv e. rr s W T2 rr s M U T2 d ev el op ed g1 48 4t Re si st an ce to A m d et ec te d N o ad di tio na l d ia gn os tic ac tio n re qu ire d A m is n ot e ff ec tiv e. rr s W T2 a nd M U T2 n o t de ve lo pe d M ut at io n in th e 14 84 r eg io n Re si st an ce to A m in fe rr ed R ec o m m en d e d : R ep ea t se co nd -li ne L PA to c on fir m th e re su lt. O p ti o n al : P er fo rm se qu en ci ng to id en tif y sp ec ifi c m ut at io n. A m is u nl ik el y to b e ef fe ct iv e. INTERPRETATION OF SECOND-LINE LINE PROBE ASSAY RESULTS Line probe assays for detection of drug-resistant tubercuLosis 18 Ta rg e t re g io n M TB D R sl p ro b e M u ta ti o n o r re g io n in te rr o ga te d In te rp re ta ti o n A d d it io n al d ia g n o st ic ac ti o n b C lin ic al im p lic at io n s ei s W T1 ei s W T1 n ot d ev el op ed M ut at io n (s ) i n th e –3 7 re gi on Re si st an ce to A m n ot de te ct ed N o ad di tio na l d ia gn os tic ac tio n re qu ire d A m is li ke ly to b e ef fe ct iv e. ei s W T2 ei s M U T1 d ev el op ed c- 14 t Re si st an ce to A m d et ec te d N o ad di tio na l d ia gn os tic ac tio n re qu ire d A m is n ot e ff ec tiv e. ei s W T2 a nd M U T1 n o t de ve lo pe d M ut at io n (s ) i n th e –1 0 to –1 4 re gi on Re si st an ce to A m n ot de te ct ed R ec o m m en d e d : R ep ea t th e se co nd -li ne L PA to co nfi rm th e re su lt. O p ti o n al : P er fo rm se qu en ci ng to id en tif y sp ec ifi c m ut at io n. c A m is li ke ly to b e ef fe ct iv e. ei s W T3 ei s W T3 n ot d ev el op ed M ut at io n (s ) i n th e –2 r eg io n N o te . N o ev id en ce th at m ut at io ns in th is r eg io n ar e as so ci at ed w ith r es is ta nc ed Re si st an ce to A m n ot de te ct ed N o ad di tio na l d ia gn os tic ac tio n re qu ire d. A m is li ke ly to b e ef fe ct iv e. a W H O n o lo ng er r ec om m en ds u se o f k an am yc in o r ca pr eo m yc in b ec au se o f t he in cr ea se d ri sk s of t re at m en t f ai lu re a nd r el ap se a ss oc ia te d w ith t he ir us e in lo ng er M D R- TB r eg im en s (1 8) . I nt er pr et at io n of se co nd -li ne L PA fo r ka na m yc in a nd c ap re om yc in is t he re fo re n ot in cl ud ed in t hi s do cu m en t. b Th e de ci si on t o pe rf or m a dd iti on al d ia gn os tic a ct io ns in di ca te d as o pt io na l s ho ul d be g ui de d by c on si de ra tio n of t he in di vi du al p at ie nt ’s r is k gr ou p fo r re si st an ce a nd b y th e pr ev al en ce o f r es is ta nc e in t he se tt in g, a s th es e fa ct or s af fe ct t he p os iti ve p re di ct iv e va lu e of t he t es t. c If se qu en ci ng r ev ea ls t he p re se nc e of t he e is m ut at io n c- 14 t, w hi ch fo r so m e re as on w as n ot d et ec te d by t he e is M U T1 p ro be , A m is n ot e ff ec tiv e. d M io tt o P, e t a l. A s ta nd ar di se d m et ho d fo r in te rp re tin g th e as so ci at io n be tw ee n m ut at io ns a nd p he no ty pi c dr ug r es is ta nc e in M yc ob ac te riu m tu be rc ul os is . E ur R es pi r J. 20 17 ;5 0 (6 ):1 70 13 54 . 19 Assessment of drug-resistant TB cases based on second-line line probe assay results ASSESSMENT OF DRUG-RESISTANT TB CASES BASED ON SECOND-LINE LINE PROBE ASSAY RESULTS Case 1. No resistance mutations detected or inferred in any of the genomic regions included in second-line line probe assay All WT bands developed, and no MUT probe bands developed in second-line LPA C on ju ga te C on tr ol A m pl ifi ca tio n C on tr ol TU B gy rA gy rA W T1 gy rA W T2 gy rA W T3 gy rA M U T1 (A 90 v ) gy rA M U T2 (S 91 P) gy rA M U T3 A (D 94 A ) gy rA M U T3 B (D 94 N /Y ) gy rA M U T3 C (D 94 G ) gy rA M U T3 D (D 94 H ) gy rB gy rB W T gy rB M U T1 (N 53 8D ) gy rB M U T2 (E 54 0v ) rr s rr s W T1 rr s W T2 rr s M U T1 (a 14 01 g) rr s M U T2 (g 14 84 t) ei s ei s W T1 ei s W T2 ei s W T3 ei s M U T1 (c -1 4t ) M Genotypic report: Resistance not detected Additional diagnostic action: Optional: • Perform phenotypic DST for Lfx at CC (e.g., CC: 1.0 mg/L in MGIT and 7H10) and for Mfx at CC and CB (e.g., CC: 0.25 mg/L in MGIT and 0.5 mg/L on 7H10; CB: 1.0 mg/L in MGIT and 2.0 mg/L on 7H10). • Perform phenotypic DST for Am if indicated. The decision to perform these optional follow-up actions should be guided by consideration of the individual patient’s risk group for resistance (e.g., prior exposure to second-line medicines, suspected treatment failure) and by the prevalence of resistance in the setting, as these factors affect the predictive value of the test. Clinical implications: Start MDR-TB treatment. Review treatment regimen according to phenotypic DST results. Line probe assays for detection of drug-resistant tubercuLosis 20 Case 2. Detection of resistance mutations associated with high-level resistance to moxiifloxacin C on ju ga te C on tr ol A m pl ifi ca tio n C on tr ol TU B gy rA gy rA W T1 gy rA W T2 gy rA W T3 gy rA M U T1 (A 90 v ) gy rA M U T2 (S 91 P) gy rA M U T3 A (D 94 A ) gy rA M U T3 B (D 94 N /Y ) gy rA M U T3 C (D 94 G ) gy rA M U T3 D (D 94 H ) gy rB gy rB W T gy rB M U T1 (N 53 8D ) gy rB M U T2 (E 54 0v ) rr s rr s W T1 rr s W T2 rr s M U T1 (a 14 01 g) rr s M U T2 (g 14 84 t) ei s ei s W T1 ei s W T2 ei s W T3 ei s M U T1 (c -1 4t ) M If one of the following MUT probes is developed: • gyrA MUT3C (i.e. gyrA D94G) (see picture above as example), • gyrA MUT3D (i.e. gyrA D94H) • gyrA MUT3B (i.e. gyrA D94N/Y) Genotypic report: Lfx: Resistance detected Mfx: Mutation associated with high-level resistance for Mfx detected Additional diagnostic action: Perform phenotypic DST for Am if indicated. Clinical implications: Mfx cannot be considered an effective medicine even at a high dose. 21 Case 3. Detection of mutations associated with at least low-level resistance to moxifloxacin SECOND-LINE LINE PROBE ASSAY RESULTS C on ju ga te C on tr ol A m pl ifi ca tio n C on tr ol TU B gy rA gy rA W T1 gy rA W T2 gy rA W T3 gy rA M U T1 (A 90 v ) gy rA M U T2 (S 91 P) gy rA M U T3 A (D 94 A ) gy rA M U T3 B (D 94 N /Y ) gy rA M U T3 C (D 94 G ) gy rA M U T3 D (D 94 H ) gy rB gy rB W T gy rB M U T1 (N 53 8D ) gy rB M U T2 (E 54 0v ) rr s rr s W T1 rr s W T2 rr s M U T1 (a 14 01 g) rr s M U T2 (g 14 84 t) ei s ei s W T1 ei s W T2 ei s W T3 ei s M U T1 (c -1 4t ) M If one of the following MUT probes is developed: • gyrA MUT1 (i.e. gyrA A90v) (see picture above as example) • gyrA MUT2 (i.e. gyrA S91P), • gyrA MUT3A (i.e. gyrA D94A), • gyrB MUT1 (i.e. gyrB N538D), • gyrB MUT2 (i.e. gyrB E540D). Genotypic report: Lfx: Resistance detected Mfx: Mutation associated with at least low-level resistance for Mfx detected Additional diagnostic action: Recommended: Perform phenotypic DST for Mfx at CB according to case 1. Perform phenotypic DST for Am if indicated. Clinical implications: Mfx could be used at a higher dose. The regimen should be re-evaluated according to phenotypic DST results at CB. Line probe assays for detection of drug-resistant tubercuLosis 22 Case 4. Precise mutation unknown, only inferred for fluoroquinolones (i.e., gyrA and gyrB) C on ju ga te C on tr ol A m pl ifi ca tio n C on tr ol TU B gy rA gy rA W T1 gy rA W T2 gy rA W T3 gy rA M U T1 (A 90 v ) gy rA M U T2 (S 91 P) gy rA M U T3 A (D 94 A ) gy rA M U T3 B (D 94 N /Y ) gy rA M U T3 C (D 94 G ) gy rA M U T3 D (D 94 H ) gy rB gy rB W T gy rB M U T1 (N 53 8D ) gy rB M U T2 (E 54 0v ) rr s rr s W T1 rr s W T2 rr s M U T1 (a 14 01 g) rr s M U T2 (g 14 84 t) ei s ei s W T1 ei s W T2 ei s W T3 ei s M U T1 (c -1 4t ) M If one of the following WT bands is not developed: • gyrA WT1 (i.e. gyrA WT1 probe missing) (see example above), • gyrA WT2 (i.e. gyrA WT2 probe missing), • gyrA WT3 (i.e. gyrA A WT3 probe missing), • gyrB WT (i.e. gyrB WT probe missing) and none of the MUT probes is developed in the gyrA and gyrB regions. Genotypic report: Lfx: Resistance inferred Mfx: Mutation associated with at least low-level resistance for Mfx inferred Additional diagnostic action: Recommended: Perform phenotypic DST for Mfx at CB according to case 1. Optional but recommended in some settings:1 gyrA and gyrB QRDR sequencing to identify resistance mutation and exclude synonymous mutations or non-synonymous mutations that do not cause resistance (systematic false-positive results) (interpret according to cases 2–4, and follow the respective recommendations for phenotypic DST). If sequencing is unavailable, perform phenotypic DST at CC for Lfx and/or Mfx as for case 1. Optional: Perform phenotypic DST for Am if indicated. 1 Lack of binding of a WT probe without simultaneous binding of a mutant probe is due to the presence of a resistance mutation (e.g., gyrA G88A). Systematic errors are possible due to synonymous or non- synonymous mutations; however, this is rare (< 1% of isolates), but these isolates may be frequent locally. Unfortunately, the settings in which these cases are frequent cannot be predicted. Thus, each laboratory must decide on the basis of local epidemiology whether sequencing of the QRDR region is necessary. For example, the gyrA A90G mutation, which prevents binding of gyrA WT2, is frequent in the Congo and the Democratic Republic of the Congo, and a synonymous mutation codon at 96 of gyrA, which prevents binding of gyrA WT3, is frequent in Medellín (Colombia) (12). In both of these settings therefore, sequencing would be recommended. 23 Clinical implications: Lfx is not effective. Mfx could be used at a higher dose. The regimen should be re- evaluated according to the phenotypic DST results at CB. Note. These recommendations do not apply if sequencing, available before treatment initiation, identifies mutations not associated with resistance to FQ or if phenotypic DST shows susceptibility at CC. Case 5. Detection of mutations that cause resistance to amikacin SECOND-LINE LINE PROBE ASSAY RESULTS C on ju ga te C on tr ol A m pl ifi ca tio n C on tr ol TU B gy rA gy rA W T1 gy rA W T2 gy rA W T3 gy rA M U T1 (A 90 v ) gy rA M U T2 (S 91 P) gy rA M U T3 A (D 94 A ) gy rA M U T3 B (D 94 N /Y ) gy rA M U T3 C (D 94 G ) gy rA M U T3 D (D 94 H ) gy rB gy rB W T gy rB M U T1 (N 53 8D ) gy rB M U T2 (E 54 0v ) rr s rr s W T1 rr s W T2 rr s M U T1 (a 14 01 g) rr s M U T2 (g 14 84 t) ei s ei s W T1 ei s W T2 ei s W T3 ei s M U T1 (c -1 4t ) M If one of the following MUT probe bands is developed: • rrs MUT1 (i.e. rrs a1401g) (see example above), • rrs MUT2 (i.e. rrs g1484t), • eis MUT1 (i.e. eis c-14t) (see example above). Genotypic report: Resistance to Am detected. Additional diagnostic action: Perform phenotypic DST for FQs according to case 1. Clinical implications: Am is not effective. Line probe assays for detection of drug-resistant tubercuLosis 24 Case 6. Precise mutation unknown, only inferred, in the rrs region If one of the following WT bands is not developed: • rrs WT1 (rrs probe WT1 not developed) (see example above), • rrs WT2 (rrs probe WT2 not developed) and none of the MUT probes is developed in the rrs region. Genotypic report: Resistance to Am inferred Additional diagnostic action: Recommended: If only the rrs WT2 probe or both rrs WT1 and WT 2 probes are not detected (and no MUT probes are detected), repeat the assay to confirm the result. Optional: Perform sequencing to identify the precise mutation. Perform phenotypic DST for FQs according to case 1. Clinical implications: Am is unlikely to be effective. C on ju ga te C on tr ol A m pl ifi ca tio n C on tr ol TU B gy rA gy rA W T1 gy rA W T2 gy rA W T3 gy rA M U T1 (A 90 v ) gy rA M U T2 (S 91 P) gy rA M U T3 A (D 94 A ) gy rA M U T3 B (D 94 N /Y ) gy rA M U T3 C (D 94 G ) gy rA M U T3 D (D 94 H ) gy rB gy rB W T gy rB M U T1 (N 53 8D ) gy rB M U T2 (E 54 0v ) rr s rr s W T1 rr s W T2 rr s M U T1 (a 14 01 g) rr s M U T2 (g 14 84 t) ei s ei s W T1 ei s W T2 ei s W T3 ei s M U T1 (c -1 4t ) M 25 Case 7. Precise mutation unknown, only inferred, in the eis region If one of the following WT bands is not developed: • eis WT1 (eis probe WT1 not developed) (e.g., eis g-37t), • eis WT2 (eis probe WT2 not developed) (e.g., eis c-12t or g-10a) (see example above), and none of the MUT probes is developed in the eis region. Genotypic report: Resistance to Am not detected (if no additional mutations in the rrs region are present) Additional diagnostic action: Recommended: Repeat the test to confirm the result. Optional: Perform phenotypic DST for FQs according to case 1. Clinical implications: Am is likely to be effective. SECOND-LINE LINE PROBE ASSAY RESULTS C on ju ga te C on tr ol A m pl ifi ca tio n C on tr ol TU B gy rA gy rA W T1 gy rA W T2 gy rA W T3 gy rA M U T1 (A 90 v ) gy rA M U T2 (S 91 P) gy rA M U T3 A (D 94 A ) gy rA M U T3 B (D 94 N /Y ) gy rA M U T3 C (D 94 G ) gy rA M U T3 D (D 94 H ) gy rB gy rB W T gy rB M U T1 (N 53 8D ) gy rB M U T2 (E 54 0v ) rr s rr s W T1 rr s W T2 rr s M U T1 (a 14 01 g) rr s M U T2 (g 14 84 t) ei s ei s W T1 ei s W T2 ei s W T3 ei s M U T1 (c -1 4t ) M 26 LINE PROBE ASSAYS FOR DETECTION OF DRUG-RESISTANT TUBERCULOSIS References 1. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance. Geneva: World Health Organization; 2021. (https://apps.who.int/iris/handle/10665/341981, accessed January 2022) 2. 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Can molecular methods detect 1% isoniazid resistance in Mycobacterium tuberculosis? J Clin Microbiol. 2013;51:1596–9. 14. Folkvardsen DB, Thomsen vØ, Rigouts L, Rasmussen EM, Bang D, Bernaerts G et al. Rifampin heteroresistance in Mycobacterium tuberculosis cultures as detected by phenotypic and genotypic drug susceptibility test methods. J Clin Microbiol. 2013;51:4220–2. 15. GenoType MTBDRsl vER 2.0. Molecular genetic assay for identification of the M. tuberculosis complex and its resistance to fluoroquinolones and aminoglycosides/ cyclic peptides from sputum specimens or cultivated samples. Instruction for use (December 2017). Nehren: Hain Lifescience; 2017. 16. Miotto P, Tessema B, Tagliani E, Chindelevitch L, Starks AM, Emerson C et al. A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis. Eur Respir J. 2017;50(6):1701354. 17. WHO consolidated guidelines on tuberculosis. Module 4: Treatment – drug-resistant tuberculosis treatment. Online annexes. Geneva: World Health Organization; 2020. (https://apps.who.int/iris/handle/10665/332397, accessed January 2022) 18. Rapid communication: Key changes to treatment of multidrug- and rifampicin-resistant tuberculosis (MDR/RR-TB). Geneva: World Health Organization; 2018 (https://apps.who.int/iris/handle/10665/275383, accessed January 2022). 19. Seifert M, Catanzaro D, Catanzaro A, Rodwell TC. Genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis: a systematic review. PLoS One. 2015;10:e0119628. 20. Kandler JL, Mercante AD, Dalton TL, Ezewudo MN, Cowan LS, Burns SP et al. validation of novel Mycobacterium tuberculosis isoniazid resistance mutations not detectable by common molecular tests. Antimicrob Agents Chemother. 2018;62(10):00974-18. 21. Andre E, Goeminne L, Cabibbe A, Beckert P, Kabamba Mukadi B, Mathys v et al. Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic mycobacteria. Clin Microbiol Infect. 2017;23:167–72. 22. Camus JC, Pryor MJ, Médigue C, Cole ST. Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology. 2002;148:2967–73. 28 LINE PROBE ASSAYS FOR DETECTION OF DRUG-RESISTANT TUBERCULOSIS Annex 1. Reporting format for first-line line probe assay results and practical examples The “Conclusion” column has been included for convenience but should not be part of the laboratory report. Example 1 Drug Gene Mutation Interpretation Conclusion Rifa rpoB H526Y Resistance to Rif detected Rif is not effective. Ha katG Mutation(s) in codon 315 region Mutation associated with high-level resistance to H inferred H is unlikely to be effective even at a high dose. inhA t-8a Eto and Pto inhA t-8a Resistance to Eto and Pto likely detected Et0 and Pto are likely not effective. Example 2 Drug Gene Mutation Interpretation Conclusion Rif rpoB No mutation detected Resistance to Rif not detected Rif is effective. Ha katG S315T Mutation associated with high-level resistance to H detected H is not effective even at a high dose.inhA c-15t Eto and Pto inhA c-15t Resistance to Eto and Pto detected Eto and Pto are not effective. Example 3 Drug Gene Mutation Interpretation Conclusion Rif rpoB Mutation(s) in codons 516–522 (435–441) Resistance to Rif inferred Rif is not effective. H katG No mutation detected Mutation likely to be associated with at least low-level resistance to H detected H at a high dose is likely to be effective. inhA t-8c Eto and Pto inhA t-8c Resistance to Eto and Pto likely detected Eto and Pto are likely not effective. a If more than one probe per drug provides information, the results should be reported according the following hierarchy (where “>”means overrule): For H: Mutation associated with high-level resistance detected > Mutation associated with high-level resistance inferred > Mutation associated with at least low-level resistance detected > Mutation associated with at least low-level resistance inferred > Resistance not detected For Rif: Resistance detected > Resistance inferred > Resistance not detected 29 ANNEx 1 Annex 2. Reporting format for second-line line probe assay results and practical examples The “Conclusion” column has been included for convenience but should not be part of the laboratory report. Example 1 Drug Gene Mutation Interpretation Conclusion Lfxa gyrA D94A Resistance to Lfx detected Lfx is not effective. Mfx could be used at higher dose. The regimen should be re-evaluated according to phenotypic DST results at CB. gyrB No mutation detected Mfxa gyrA D94A Mutation associated with at least low-level resistance to Mfx detected gyrB No mutation detected Ama rrs a1401g Resistance to Am detected Am is not effective. eis promoter No mutation detected Example 2 Drug Gene Mutation Interpretation Conclusion Lfx gyrA A90v Resistance to Lfx detected Lfx is not effective. Mfx could be used at a higher dose. The regimen should be re-evaluated according to phenotypic DST results at CB. gyrB No mutation detected Mfx gyrA A90v Mutation associated with at least low-level resistance to Mfx detected gyrB No mutation detected Am rrs No mutation detected Resistance to Am detected Am is not effective. eis promoter c-14t 30 LINE PROBE ASSAYS FOR DETECTION OF DRUG-RESISTANT TUBERCULOSIS Example 3 Drug Gene Mutation Interpretation Conclusion Lfx gyrA Mutation(s) in codons 89-93 Resistance to Lfx inferred Lfx is not effective. Mfx could be used at a higher dose. The regimen should be re-evaluated according to phenotypic DST results at CB. Note. These recommendations do not apply if sequencing, available before treatment initiation, identifies mutations not associated with resistance to FQ, or if phenotypic DST shows susceptibility at CC. gyrB No mutation detected Mfx gyrA Mutation(s) in codons 89-93 Mutation(s) associated with at least low-level resistance to Mfx inferred gyrB No mutation detected Am rrs No mutation detected Resistance to Am not detected Am is effective. eis promoter Mutation(s) in the –37 region a If more than one probe per drug provides information, the results should be reported according to the following hierarchy (where “>”means overrule): For Lfx and Am: Resistance detected > Resistance inferred > Resistance not detected For Mfx: Mutation associated with high-level resistance detected > Mutation associated with at least low-level resistance detected > Mutation associated with at least low-level resistance inferred > Resistance not detected 31 Annex 3. Specific nucleotide changes detected with mutation probes ANNEx 2 Some of the amino acid (AA) changes identified with first- and second-line LPAs are due to nucleotide changes that are not specifically recognized by the MUT probes. For instance, the gyrA mutation A90v is due to two possible nucleotide changes: gcg > gtg or gcg>gtc. However, only the first, gcg > gtg, will be recognized by the gyrA MUT1 probe, while the second, gcg > gtc will be detected only by the absence of gyrA WT2 (i.e., gyrA WT2 not detected). MUT probe AA change Nucleotide change rpoB MUT probes MUT1 D516v (D435v) gac > gtc MUT2A H526Y (H445Y) cac > tac MUT2B H526D (H445D) cac > gac MUT3 S531L (S450L) tcg > ttg MUT probe AA change Nucleotide change katG MUT probes MUT1 S315T agc>acc MUT2 S315T agc>aca MUT probe AA change Nucleotide change gyrA MUT probes MUT1 A90v gcg>gtg MUT2 S91P tcg>ccg MUT3A D94A gac>gcc MUT3B D94N gac>aac MUT3B D94Y gac>tac MUT3C D94G gac>ggc MUT3D D94H gac>cac MUT probe AA change Nucleotide change gyrB MUT probes MUT1 N538D (N499D) aac > gac MUT2 E540v (E501v) gaa > gta For further information, please contact: Global TB Programme World Health Organization 20, Avenue Appia CH-1211 Geneva 27 Switzerland Web site: www.who.int/tb