Supplemental Table 1. Country of origin of the samples collected for the studies included in this systematic review. Authors PubMed ID.

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Benjamin Cain
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1 Supplemental Note In order to obtain all articles relevant to this study, key search terms were used in various combinations with the AND operator. Search terms included: pyrazinamide, resistance, Mycobacterium tuberculosis, pnca, mutations, and pza. Titles and abstracts were read and if the article seemed to include all necessary inclusion criteria based solely on the title and abstract, the entire article was read. Importantly, once an article was found that met inclusion criteria, the references were analysed for other potential articles. The same procedure was used for articles found in references of relevant papers as those found through a search in PubMed. Supplemental Figure 1. Number of pyrazinamide-resistant Mycobacterium tuberculosis isolates with each type of mutation in pnca open reading frame or its promoter (Note: Figure only reports consequential mutations, i.e. does not include synonymous mutations in pnca open reading frame.) Notation: SUB = Isolates harboring a single point (includes synonymous and nonsynonymous changes); DEL = Single deletion (of varying length); INS = Single insertion of varying length; numerals: Indicate the number of distinct mutations of a certain type (e.g. 3 SUBs = three distinct substitutions in a single isolate); + : indicates the combination of more than one type of mutation in an isolate (e.g. 2 SUBs + DEL = all isolates harboring two distinct consequential substitutions and one deletion); Supplemental Table 1. Country of origin of the samples collected for the studies included in this systematic review. Authors PubMed ID Published Date Geographic Location Number Resistant Clinical Sequenced Number Susceptible Clinical Sequenced Alexander et al *not noted* Ando et al Japan Aono et al Japan Aragon et al Spain

2 Barco et al Brazil Bishop et al Gauteng, South Africa Brown et al Turkey, England Cheng et al *not noted* Canada, United States, South Korea Chiu et al Taiwan Clemente et al Brazil Cuevas-Cordoba Mexico et al. Daum et al South Africa Davies et al South Africa Doustar et al Iran Endoh et al Japan Feuerriegel et al Sierra Leone Hannan et al United States Hirano et al Myanmar, Thailand, Yemen, Nepal, India, Korea, Indonesia, Malaysia, Phillipines, Bangladesh, Canada Hoffner et al *not noted* Hou et al China Huang et al Taiwan Huang et al China Jnawali et al South Korea Jonmalung et al Thailand Jureen et al *not noted* Lee et al Singapore Lemaitre et al France Marttila et al Russia McCammon et al United States, Mexico Mestdagh et al Bangladesh, Azerbaijan, Siberia, Belgium, Abkhazia, Rwanda, Congo- Brazzaville, Kazakhstan, Canada, Scotland Miotto et al Various locations not noted Miyagi et al Japan Morlock et al United States

3 Mphalele et al South Africa Muthaiah et al India Pandey et al New Zealand Park et al Korea Pholwat et a Thailand, Tanzania, United States Portugal et al Portugal Rodrigues et al Brazil Sachais et al United States Scorpio & United States Zhang Scorpio et al *not noted* Sekiguchi et al Japan, Poland Sekiguchi et al Japan, Poland Sheen et al Sheen et al Peru Shenai et al India Simons et al Netherlands Somoskovi et al *not noted* Sreevatsan et al *not noted* Stoffels et al Belgium, Georgia, Chechnia, Rwanda Suzuki et al Japan Therese et al Tracevska et al Latvia Wade et al Canada, United States, South Korea Watcharasamph *not noted* ankul et al. Werngern et al Sweden Yuksel et al Turkey Zhang et al China Zhou et al China Supplemental Table 2. List of mutations discovered in PZase or the pnca promoter region of PZA R Mtb isolates with varying pyrazinamide MIC Levels. Multiple Lines for a Mutation Indicate Differing MIC Levels Reported by Different Studies. Mutation Number Resistant MIC Level -16AACGTA-11--> GGCAGTT A-11G A-11G 1 300

4 T-7C bp DEL bp DEL Met1Ile 3 > bp DEL at Start 2 >900 Ala3Pro Ala3Pro Ala3Glu Ala3Glu 1 >400 Leu4Ser Leu4Ser 1 >400 C INS Ile5Asn 1 >400 Ile5Ser 1 >400 Ile5Ser 1 >900 Ile6Thr Ile6Thr 1 >100 <300 Val7Ala Val7Gly Val9Gly Gln10Arg Gln10Pro C DEL Gln10Pro Gln10Pro 2 >1600 Gln10Pro 1 >400 Gln10STOP 1 >800 C DEL 28 1 >900 Asp12Ala Asp12Gly Asp12Ala Asp12Ala 1 >100 Asp12Ala 1 >300 Asp12Asn 1 >900 Phe13Ser 1 >2000 Cys14Arg 1 >800 Gly17Asp 2 >800 G INS 52 1 >100 2bp INS 54 1 >400 Leu19Pro 1 600

5 C DEL C DEL 59 2 >1600 Gly24Asp G DEL G DEL 71 3 >800 G DEL 71 1 >900 Ala26Gly 1 >900 Tyr34Asp Tyr34Asp Tyr34STOP Tyr34Asp 2 >400 Tyr34Ser 2 >800 C DEL >900 CG INS >100 < kb DEL Tyr41His Tyr41STOP Tyr41STOP 1 >800 CG DEL His43Pro 1 >800 Ala46Val 6 >300 Ala46Glu 1 >300 Ala46Val 1 >800 G INS >800 Thr47Ala 1 64 Thr47Pro Lys48Thr 1 49 Asp49Asn His51Pro His51Gln His51Gln 1 >1024 His51Arg 1 >800 His51Arg 1 >800 His51Pro 1 >800 His51Arg 1 >800 His51Gln 1 >900 Asp53Ala 1 >300 Asp63Gly 1 >800 Pro54Leu Pro54Thr 1 900

6 Pro54Ser 1 >1024 Pro54Leu 2 >1600 Pro54Arg 1 >400 G DEL >500 Pro54Gln 1 >800 Pro54Thr 1 >900 His57Asp 1 >400 His57Arg 1 >800 Phe58Leu Phe58Leu Phe58Leu 1 >800 Ser59Pro Ser59Pro 1 >800 Thr61Pro Pro62Gln Pro62Leu 2 >400 Pro62Arg 1 >800 2bp INS >400 Asp63His 1 >500 A INS >300 Ser67Pro 1 >800 Trp68STOP 1 >300 Trp68Leu 2 >800 Pro69Arg Pro69Leu 1 >2000 Pro69Arg 2 >300 His71Tyr His71Arg 1 >1600 His71Glu 1 >300 His71Arg 1 >800 Cys72Arg bp INS >2000 Cys72Arg 1 >800 Cys72Arg 1 >900 Thr76Pro Thr76Pro 1 >800 Thr76Pro 1 >800 Thr76Pro 1 >900 Gly78Cys Gly78Asp 1 >800 His82Arg 1 >500

7 Leu85Pro Leu85Arg 1 >400 Leu85Arg 1 >500 Leu85Pro 2 >800 Leu85Pro 3 >900 Thr87Met AC INS bp INS >400 Ser88STOP Phe94Leu Phe94Ser 1 >300 Lys96Thr/G DEL Lys96Asn Lys96Gln 1 >1600 G DEL >400 G DEL >500 Lys96Thr 1 >800 T INS >800 Lys96Asn 1 >900 Gly97Asp Gly97Ser Gly97Asp Gly97Asp 1 >300 Gly97Asp 1 >400 Gly97Ser 2 >500 Gly97Cys 1 >900 Tyr99STOP 1 >800 Tyr99STOP 1 >800 A INS > G DEL >300 1 >500 Tyr103STOP C INS Tyr103Cys Ser104Arg 1 >800 Gly105Asp 2 >800 C INS >800 Gly108Arg 1 > bp INS G DEL >500 Thr114Pro 1 >800 Leu116Pro 1 100

8 Asn118Thr bp DEL >300 Leu120Arg 1 >800 Arg121Pro bp INS >1600 Gln122STOP 1 >900 Arg123Pro AG INS Arg123Leu 1 >900 Val125Phe 1 > bp DEL >1600 8bp DEL >400 Val130Gly/GG INS >500 Val130Gly 1 >800 9bp INS 388 (>800) 1 >800 Val130Gly 1 >800 GG INS G INS >1600 GG INS >1600 G DEL >900 Gly132Ala Gly132Asp Gly132Ser Gly132Asp 1 >800 17bp DEL >800 Gly132Ser 2 >900 Ile133Asn G INS >300 Ile133Ser 1 >800 TT DEL >900 Ala134Val 1 >500 Ala134Val 1 >800 CC INS Thr135Pro 1 >800 Thr135Pro 1 >800 His137Pro 1 >1600 His137Arg 2 >800 His137Pro 1 >900 Cys138Tyr 1 >2000 Cys138Arg 1 >400 Cys138Ser 1 >500

9 Cys138Ser 1 >500 Cys138Tyr 1 >800 Cys138Tyr 1 >900 Val139Leu Val139Leu 1 >2000 Val139Ala 2 >2000 Val139Gly 1 >800 Val139Ala 1 >800 Val139Leu 2 >900 TG DEL >900 Val139Met 1 >900 68bp DEL GC INS >900 Arg140Ser/8bp DEL >900 G INS >900 Gln141Pro 1 >1600 Gln141Pro 1 >500 Gln141Pro 1 >900 Gln141Pro 1 >900 Thr142Lys Thr142Lys 1 >1024 Thr142Ala 1 >800 Thr142Lys 2 >800 Thr142Lys 1 >900 Thr142Met 1 >900 GG INS >900 Ala146Val 1 >800 G DEL >900 8-bpDEL >900 Thr153Asn Val155Gly Val155Gly Val155Gly 1 >1024 Val155Gly 2 >300 Val155Glu 1 >800 Val155Gly 1 >900 C INS >900 9bp INS 484/2bp DEL >400 Ser164Pro 1 >800 C INS >800 1bp INS >400

10 CG INS > Thr168Asn 1 >900 Ala171Thr 1 >800 Ala171Pro 1 >900 Leu172Pro Leu172Pro Leu172Pro 1 >800 5-bp INS >900 G INS >1600 Met175Thr 1 >1024 A INS Leu182Ser AG DEL 94/GA DEL 97 1 >1600 T-7C/Thr114Ala 2 >1600 Supplemental Table 3. List of media and concentrations used for pyrazinamide drug susceptibility testing for each study included in this systematic review. Authors PubMed ID Published Date DST/MIC (Media used) Alexander et al DST (BACTEC (BACTEC 460TB) Ando et al (BACTEC Aono et al DST (BACTEC MIC (BACTEC Aragon et al DST (BACTEC 460) (BacT/ALERT PZA kit) Barco et al MIC (MABA) (LJ Proportion) Bishop et al MIC (BACTEC 460TB) Brown et al DST (BACTEC 460TB Concentration(s) (Susceptibility Cutoff) *not noted* (usually 100mg/L) *not noted* (usually 100mg/L) 100 ug/ml 200, 300, 400, 800, 1600 ug/ml *not noted* mg/L mg/L (100 mg/l) (100mg/L) (100mg/L)

11 Cheng et al MIC (BACTEC 460TB) (Lowenstein- Jensen) (100mg/L), 300, ,500mg/L Chiu et al DST (100mg/L) (BACTECT Clemente et al DST (BACTEC 460) *not noted* Cuevas-Cordoba et al DST (BACTECT (100mg/L) Daum et al DST (BACTEC *not noted* (usually 100mg/L) Davies et al DST (BACTEC (100mg/L) 460TB) Doustar et al DST (7H9 broth) (100mg/L) Endoh et al MIC (7H10) 25mg/l, 50mg/l, (100 mg/l) (7H9) Feuerriegel et al DST (BACTEC 460) MIC (BACTEC Hannan et al DST (BACTEC 460TB) Hirano et al MIC (Middlebrook 7H11) Hoffner et al DST (BACTEC MIC (BACTEC Hou et al DST MIC (BACTEC 460TB) (Proportion Method) Huang et al MIC (BACTEC 100mg/L, 400mg/L (100mg/L) 100, 50, 25, 12.5, 6.3mg/L (100mg/L) mg/L 100ug/ml ug/ml (100mg/L),300, 400, 500, 600, 800, 900, 1000, 1200, 1500, mg/L (100mg/L), 300mg/L Huang et al DST (LJ) (100mg/L) Jnawali et al DST (Proportion (100mg/L) Method) Jonmalung et al DST (BACTEC (100mg/L) Jureen et al DST (BACTEC (100mg/L) 460) Lee et al DST (BACTEC *not noted* (usually

12 460) 100mg/L) Lemaitre et al MIC (7H9 (500mg/L),50mg/L liquid) Marttila et al DST (BACTEC (100mg/L) 460TB) McCammon et al DST (100mg/L) (BACTEC460) Mestdagh et al DST (BACTEC (100mg/L) 460TB) Miotto et al DST (BACTEC "according to manufacturer's instructions" = 100mg/L Miyagi et al MIC (7H9 broth) (100), 200, 250, 400mg/L Morlock et al DST (7H10 agar) (25mg/L) (100mg/L) MIC (BACTEC 460TB) Mphalele et al DST (BACTEC (100mg/L) 460) Muthaiah et al DST (Pportion (300mg/L) Mhod) Pandey et al DST (BACTEC (100mg/L) (BACTEC 460TB) Park et al DST (Pportion (100mg/L), 500mg/L Mhod) Pholwat et a DST (BACTEC (100mg/L) Portugal et al DST (BACTEC (100mg/L) Rodrigues et al DST(Pportion *not noted* Mod) Sachais et al DST (BACTEC 460TB) Scorpio & Zhang MIC (7H9 liquid) Scorpio et al MIC (BACTEC 460TB) Sekiguchi et al DST (BACTEC Sekiguchi et al DST (BACTEC Sheen et al Sheen et al DST (BACTEC 460TB) (7H9 broth) *not noted* (usually 100mg/L) 50,(100mg/L),200,500,1000 (100mg/L), 300mg/L, 900mg/L *not noted* (usually 100mg/L) *not noted* (usually 100mg/L) (100mg/L) (200mg/L)

13 Shenai et al DST (BACTEC (100mg/L) Simons et al DST (BACTECT *not noted* (usually 100mg/L) Somoskovi et al DST (BACTEC (100mg/L) 460) Sreevatsan et al DST (7H10 agar) (BACTEC (25mg/L) *not noted* (usually 100mg/L) 460TB) Stoffels et al DST (BACTEC *not noted* (usually 100mg/L) MIC (BACTEC Suzuki et al DST (7H10) (100mg/L) Therese et al DST (BACTEC (100mg/L) 460TB) Tracevska et al DST (BACTEC) *not noted* (usually 100mg/L) Wade et al MIC (100mg/L) (determined in Cheng et al, Scorpio et al.) Watcharasamphankul DST (BACTEC (100mg/L) et al. MGIT) Werngern et al DST (BACTEC 460) (100mg/L) OR MIC (BACTEC Yuksel et al DST (BACTEC 460) *not noted* (usually 100mg/L) Zhang et al DST & MIC 25, 50, (100), (200), 400, Zhou et al DST (BACTEC 800mg/L (100mg/L) Supplemental Table 4. PRISMA checklist Section/topic # Checklist item TITLE Reported on page #

14 Title 1 Identify the report as a systematic review, Meta-analysis, or both. ABSTRACT Structured summary 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis Methods; results; limitations; conclusions and implications of key findings; systematic review registration number. INTRODUCTION Rationale 3 Describe the rationale for the review in the context of what is already known. Objectives 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). MHODS Protocol and registration 5 Indicate if a review Protocol exists, if and where it can be accessed (e.g., Web address), and, if available, Provide registration information including registration number. Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of followup) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. Study selection 9 State the Process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the Ma-analysis). Data collection Process 10 Describe Method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any Processes for obtaining and confirming data from investigators. Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made. Risk of bias in individual studies 12 Describe Methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means). Synthesis of results 14 Describe the Methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I 2 ) for each Ma-analysis. Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies) , , 14-15

15 Additional analyses 16 Describe Mhods of additional analyses (e.g., sensitivity or subgroup analyses, Ma-regression), if done, indicating which were pre-specified. RESULTS Study selection 17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and Provide the citations. Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot. Synthesis of results 21 Present results of each Ma-analysis done, including confidence intervals and measures of consistency. Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15). Additional analysis 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, Ma-regression [see Item 16]). DISCUSSION Summary of evidence 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare Providers, users, and policy makers). Limitations 25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias). Conclusions 26 Provide a general interpretation of the results in the context of other evidence, and implications for future research. FUNDING Funding 27 Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review ,

16 Supplemental Table 5. Sensitivity and specificity calculated based on four different criteria for variants associated with PZA resistance in Mycobacterium tuberculosis clinical isolates. Resistance Association Criteria Sensitivity Specificity All mutations observed in PZA resistant isolates are associated with PZA resistance 83% 91% Only mutations that exclusively appear in resistant isolates (never appear in susceptible isolates) are associated with resistance 59% 100% Consider mutations associated with resistance 70% of the time (at least 70% of isolates that harbour these mutations are resistant) 63% 94% Consider mutations associated with resistance 60% of the time (at least 70% of isolates that harbour these mutations are resistant) 70% 91% Consider mutations associated with resistance 50% of the time (at least 70% of isolates that harbour these mutations are resistant) 78% 84% Supplemental Table 6. Statistical analysis of all resistance Associated mutations published in the included literature for diagnostic testing. Positive coordinates are amino acid numbers in PZase while negative coordinates indicate the position of the mutation in the promoter of pnca. Codon Mutation -16AACGTA-11--> GGCAGTT Number of Isolates Sensitivity Specificity Positive Predicti ve Value Negative Predicti ve Value % 100% 100% 55% G-13T % 100% 100% 55% T DEL % 100% 100% 55% T-12G % 100% 100% 55% T-12C % 100% 100% 55% A-11G % 100% 90% 55% A-11C % 100% 50% 55% A % 100% 100% 55% T-10C % 100% 100% 55% T-7C % 100% 100% 55% G DEL % 100% 100% 55% C INS % 100% 50% 55% 11bp DEL % 100% 100% 55% 8bp DEL % 100% 100% 55% Met1Ile % 100% 83% 55% Met1Lys % 100% 100% 55% Met1Thr % 100% 100% 55% 11bp DEL START % 100% 100% 55% Ala3Glu % 100% 100% 55% Ala3Pro % 100% 100% 55% Ala3Ser % 100% 100% 55% Ala3Gln % 100% 100% 55% Leu4Ser % 100% 100% 55%

17 Leu4Trp % 100% 100% 55% 6bp DEL % 100% 100% 55% C INS % 100% 100% 55% Ile5Asn % 100% 100% 55% Ile5Ser % 100% 100% 55% C INS % 100% 100% 55% Ile6Thr % 100% 100% 55% Ile6Ser % 100% 100% 55% G INS % 100% 100% 55% Ile6Leu % 96% 6% 54% Val7Ala % 100% 100% 55% Val7Gly % 100% 92% 55% Val7Phe % 100% 100% 55% Asp8Gly % 100% 100% 55% Asp8Asn % 100% 100% 55% Asp8Tyr % 100% 100% 55% Asp8Ala % 100% 100% 55% Asp8Glu % 100% 100% 55% TCG INS % 100% 100% 55% Val9Gly % 100% 100% 55% Gln10Pro % 100% 100% 55% Gln10Lys % 100% 100% 55% Gln10STOP % 100% 100% 55% Gln10Arg % 100% 89% 55% C DEL % 100% 100% 55% IS6110 INS % 100% 100% 55% Asp12Ala % 100% 91% 55% Asp12Asn % 100% 100% 55% Ala12Gly % 100% 100% 55% Asp12Glu % 100% 100% 55% Asp12Gly % 100% 93% 55% G INS % 100% 100% 55% Phe13Ser % 100% 67% 55% GACT INS % 100% 100% 55% Phe13Leu % 100% 75% 55% Cys14Arg % 100% 93% 55% Cys14STOP % 100% 100% 55% Cys14Trp % 100% 100% 55% Cys14Tyr % 100% 100% 55% Cys14Gly % 100% 40% 55% C INS % 100% 100% 55% 11bp DEL % 100% 100% 55% Gly16Ser % 100% 100% 55%

18 Gly17Asp % 100% 100% 55% Gly17Ser % 100% 75% 55% G DEL % 100% 100% 55% C INS % 100% 100% 55% G INS % 100% 100% 55% Met175Ile % 100% 85% 55% G INS % 100% 100% 55% 2bp INS % 100% 100% 55% Ser18Pro % 100% 100% 55% Val188Leu % 100% 100% 55% Leu19Pro % 100% 100% 55% Leu19Arg % 100% 100% 55% 234bp DEL % 100% 100% 55% C DEL % 100% 100% 55% Ala20STOP % 100% 100% 55% G DEL % 100% 100% 55% Val21Ala % 100% 25% 55% Val21Gly % 100% 100% 55% G DEL % 100% 100% 55% A DEL % 100% 100% 55% Gly23Ala % 100% 100% 55% G DEL % 100% 100% 55% G DEL % 100% 100% 55% Gly24Asp % 100% 100% 55% G DEL % 100% 100% 55% C DEL % 100% 100% 55% 5bp DEL % 100% 100% 55% Ala26Gly % 100% 100% 55% GC INS % 100% 100% 55% G INS codon % 100% 100% 55% Leu27Pro % 100% 88% 55% Ala28Val % 100% 100% 55% C DEL % 100% #DIV/0! 55% C DEL % 100% 100% 55% C INS % 100% 100% 55% 24bp DEL % 100% 100% 55% Ile31Ser % 100% 57% 55% lle31thr % 100% 100% 55% Ile31Thr % 100% 100% 55% Asp33Ala % 100% 50% 55% C DEL % 100% 100% 55% Tyr34Asp % 100% 100% 55% Tyr34STOP % 100% 100% 55%

19 Tyr34Ser % 100% 100% 55% A INS % 100% 100% 55% Tyr34Cys % 100% 100% 55% 167bp DEL % 100% 100% 55% C DEL % 100% 100% 55% Leu35Pro % 100% 89% 55% Leu35Arg % 100% 33% 55% C DEL % 100% 100% 55% G INS codon % 100% 100% 55% CG INS % 100% 100% 55% C DEL % 100% 100% 55% 5.3kb DEL % 100% #DIV/0! 55% Ala36Ala % 100% 100% 55% Glu37stop % 100% 100% 55% Tyr41STOP % 100% 100% 55% Tyr41His % 100% 100% 55% T INS % 100% 100% 55% CG DEL % 100% 100% 55% His43Pro % 100% 100% 55% Val44Gly % 100% 100% 55% GG INS % 100% 100% 55% Val45Ala % 100% 75% 55% Val45Gly % 100% 100% 55% Ala46Val % 100% 80% 55% Ala46Pro % 100% 100% 55% Ala46Glu % 100% 100% 55% Val46Asn % 100% 100% 55% G INS % 100% 100% 55% Ala46Ser % 100% 100% 55% 7bp INS % 100% 100% 55% Thr47Ala % 100% 67% 55% Thr47Pro % 100% 100% 55% Thr47Ser % 100% 100% 55% Lys48Glu % 100% 60% 55% Lys48stop % 100% 100% 55% Lys48Thr % 100% 100% 55% Lys48Arg % 100% 100% 55% Asp49Gly % 100% 100% 55% Asp49Ala % 100% 100% 55% Asp49Asn % 100% 100% 55% Asp49Val % 100% 100% 55% Asp49Tyr % 100% 100% 55% A DEL % 100% 100% 55%

20 80bp DEL % 100% 100% 55% G INS % 100% 100% 55% T DEL % 100% 100% 55% His51Arg % 100% 92% 55% His51Asp % 100% 100% 55% His51Gln % 100% 100% 55% T INS % 100% 100% 55% His51Pro % 100% 91% 55% His51Tyr % 100% 100% 55% His51Asn % 100% 100% 55% His51Leu % 100% 100% 55% Asp53Ala % 100% 100% 55% Asp53Asn % 100% 100% 55% 44bp DEL % 100% 100% 55% Pro54Arg % 100% 100% 55% Pro54Leu % 100% 93% 55% Pro54Gln % 100% 88% 55% Pro54Thr % 100% 100% 55% Pro54Ser % 100% 100% 55% G DEL % 100% 100% 55% G DEL % 100% 100% 55% C INS % 100% 100% 55% His57Arg % 100% 100% 55% His57Asp % 100% 100% 55% His57Pro % 100% 100% 55% T INS % 100% 100% 55% His57Gln % 100% 100% 55% His57Tyr % 100% 88% 55% Phe58Leu % 100% 81% 55% T INS % 100% 100% 55% Phe58Ser % 100% 100% 55% Ser59Pro % 100% 100% 55% Thr61Pro % 100% 100% 55% 5bp INS % 100% 100% 55% 2bp DEL % 100% 100% 55% Pro62Gln % 100% 100% 55% Pro62Leu % 100% 100% 55% Pro62His % 100% 100% 55% 4bp INS % 100% 100% 55% A INS % 100% 100% 55% C DEL % 100% 100% 55% Pro62Arg % 100% 100% 55% Pro62Thr % 100% 100% 55%

21 T INS % 100% 100% 55% Asp63Gly % 100% 100% 55% Asp63Ala % 100% 100% 55% 2bp INS % 100% 100% 55% T INS % 100% 100% 55% Asp63His % 100% 100% 55% GG INS % 100% 100% 55% A INS % 100% 100% 55% Tyr64stop % 100% 100% 55% Tyr64Asp % 100% 100% 55% Ser65Ser % 99% 36% 55% A INS % 100% 100% 55% 7bp ins % 100% 100% 55% Ser65Pro % 100% 33% 55% Ser65Ser/Frameshift % 100% 100% 55% 14BP DEL % 100% 100% 55% Ser66Pro % 100% 100% 55% Ser67Pro % 100% 100% 55% 8bp INS % 100% 100% 55% Trp68Leu % 100% 100% 55% Trp68STOP % 100% 100% 55% Trp68Gly % 100% 94% 55% Trp68Arg % 100% 93% 55% Trp68Cys % 100% 100% 55% Pro69Arg % 100% 100% 55% Pro69Leu % 100% 100% 55% His71Tyr % 100% 100% 55% His71Arg % 100% 75% 55% His71Glu % 100% 100% 55% His71Asn % 100% 100% 55% 14BP INS % 100% 100% 55% 3bp DEL % 100% 100% 55% His71Asp % 100% 100% 55% His71Gln % 100% 100% 55% His71Thr % 100% 100% 55% T DEL % 100% 100% 55% His71Pro % 100% 100% 55% Cys72Arg % 100% 100% 55% Cys72STOP % 100% 100% 55% Cys72Tyr % 100% 100% 55% 29bp INS % 100% 100% 55% Cys72Trp % 100% 100% 55%

22 10bp INS % 100% 100% 55% G INS % 100% 100% 55% G INS % 100% 100% 55% Thr76Ile % 100% 100% 55% Thr76Pro % 100% 98% 55% Thr76Ala % 100% 100% 55% 251bp DEL % 100% 100% 55% IS 6110 INS % 100% 100% 55% C DEL % 100% 100% 55% 4bp INS % 100% 100% 55% G INS % 100% 100% 55% Gly78Asp % 100% 100% 55% Gly78Cys % 100% 100% 55% Gly78Val % 100% 100% 55% Gly78Gly % 100% 100% 55% A DEL % 100% 100% 55% Phe81Val % 100% 67% 55% Phe81Cys % 100% 100% 55% His82Arg % 100% 100% 55% His82Asp % 100% 67% 55% His82Leu % 100% 100% 55% CA INS % 100% 100% 55% Pro83Arg % 100% 100% 55% C INS % 100% 100% 55% Leu85Gln % 100% 100% 55% Leu85Arg % 100% 100% 55% Leu85Pro % 100% 98% 55% 14bp DEL % 100% 100% 55% GG INS % 100% 100% 55% 18bp INS % 100% 100% 55% 1bp DEL % 100% 100% 55% AC INS % 100% 100% 55% Thr87Met % 100% 100% 55% 2bp INS % 100% 100% 55% AC INS % 100% 100% 55% 68bp DEL % 100% 100% 55% Ser88STOP % 100% 100% 55% Ile90Ser % 100% 100% 55% Glu91STOP % 100% 100% 55% Val93Leu % 100% 100% 55% 106bp INS % 100% 100% 55% Phe94Ser % 100% 100% 55% Phe94Cys % 100% 83% 55%

23 Phe94Leu % 100% 93% 55% Phe94Pro % 100% 100% 55% Tyr95stop % 100% 100% 55% CA DEL % 100% 100% 55% Lys96Gln % 100% 100% 55% Lys96Thr % 100% 100% 55% Lys96Arg % 100% 100% 55% G DEL % 100% 100% 55% T INS % 100% 100% 55% Lys96Asn % 100% 100% 55% A DEL % 100% 100% 55% Lys96Glu % 100% 93% 55% 33bp INS % 100% 100% 55% T INS % 100% 100% 55% Lys96STOP % 100% 100% 55% Gly97Asp % 100% 93% 55% Gly97Cys % 100% 100% 55% Gly97Ser % 100% 100% 55% Gly97Ala % 100% 100% 55% T DEL % 100% 100% 55% G DEL % 100% 100% 55% T INS % 100% 100% 55% Gly97Arg % 100% 100% 55% 10bp DEL % 100% 100% 55% Tyr99STOP % 100% 100% 55% Thr100Pro % 100% 100% 55% Thr100Ala % 100% 100% 55% G DEL % 100% 100% 55% A INS % 100% 100% 55% A DEL % 100% 100% 55% Ala102Val % 100% 56% 55% Ala102Thr % 100% 100% 55% Als102Val % 100% 100% 55% Tyr103Asp % 100% 100% 55% Tyr103Cys % 100% 75% 55% Tyr103His % 100% 100% 55% C INS % 100% 100% 55% Tyr103STOP % 100% 95% 55% Tyr103Ser % 100% 100% 55% Ser104Cys % 100% 100% 55% Ser104Arg % 100% 100% 55% Ser104Ile % 100% 100% 55% C INS % 100% 100% 55%

24 Gly105Asp % 100% 100% 55% G INS % 100% 100% 55% Gly105Val % 100% 100% 55% CT INS % 100% 100% 55% Glu107STOP % 100% 100% 55% Gly108Arg % 100% 100% 55% Gly108STOP % 100% 100% 55% Gly108Val % 100% 100% 55% A DEL % 100% 100% 55% Glu111STOP % 100% 100% 55% Asn112Tyr % 100% 100% 55% G INS % 100% 100% 55% G DEL % 100% 100% 55% G DEL % 100% 100% 55% Thr114Pro % 100% 100% 55% Tyr114Ser % 100% 100% 55% 1355bp INS % 100% 100% 55% IS6110 INS codon % 100% 100% 55% C DEL % 100% 100% 55% Leu116Pro % 100% 100% 55% Leu116Arg % 100% 100% 55% Leu116Gly % 100% 100% 55% C INS % 100% 100% 55% T INS % 100% 100% 55% Asn118Thr % 100% 100% 55% 7bp DEL % 100% 100% 55% Trp119Arg % 100% 100% 55% Trp119Cys % 100% 67% 55% Trp119Gly % 100% 100% 55% Trp119Ser % 100% 100% 55% Trp119Stop % 100% 100% 55% Trp119Leu % 100% 100% 55% Leu120Arg % 100% 100% 55% Leu120Pro % 100% 100% 55% Leu120Gln % 100% 100% 55% 81bp DEL % 100% 100% 55% C DEL % 100% 100% 55% Arg121Pro % 100% 100% 55% 8bp INS % 100% 100% 55% Gln122STOP % 100% 100% 55% Gln122Gln % 100% 100% 55% C INS % 100% 100% 55%

25 Arg123Leu % 100% 100% 55% Arg123Pro % 100% 100% 55% AG INS % 100% 100% 55% 16bp DEL % 100% 100% 55% 14bp DEL % 100% 100% 55% C INS % 100% 100% 55% Val125Phe % 100% 100% 55% 9BP INS % 100% 100% 55% Val125Asp % 100% 100% 55% 15bp DEL % 100% 100% 55% Val125Gly % 100% 100% 55% Val125Leu % 100% 100% 55% GA INS % 100% 100% 55% 10bp DEL % 100% 100% 55% 30bp DEL % 100% 100% 55% 9bp DEL % 100% 100% 55% Glu127Lys % 100% 100% 55% Glu127Cys % 100% 100% 55% 11BP DEL % 100% 100% 55% Glu127STOP % 100% 100% 55% 8bp DEL % 100% 100% 55% Val128Gly % 100% 94% 55% AG INS % 100% 100% 55% 9bp DEL % 100% 100% 55% 9bp DEL codon % 100% 100% 55% 5BP DEL % 100% 100% 55% Asp129Tyr % 100% 100% 55% 3bp DEL % 100% 100% 55% CG INS % 100% 100% 55% 11-bpDEL % 100% 100% 55% GG INS % 100% 100% 55% Val130Gly % 100% 100% 55% Val130Ala % 100% 80% 55% GG INS % 100% 100% 55% 9bp INS % 100% #DIV/0! 55% G INS % 100% 100% 55% GG INS codon % 100% 100% 55% 9-bp INS % 100% 100% 55% GG INS % 100% 100% 55% G INS % 100% 100% 55% G INS % 100% 100% 55% GG INS % 100% 100% 55% Val131Phe % 100% 100% 55%

26 G INS % 100% 100% 55% GG INS % 100% 100% 55% GT INS % 100% 100% 55% T INS % 100% 100% 55% G DEL % 100% 100% 55% 9bp INS % 100% 100% 55% Gly132Asp % 100% 100% 55% Gly132Ser % 100% 100% 55% Gly132Val % 100% 100% 55% 17bp DEL % 100% 100% 55% Gly132Ala % 100% 100% 55% 6bp INS % 100% 100% 55% Gly132Cys % 100% 100% 55% Gly132STOP % 100% 100% 55% Ile133Ser % 100% 100% 55% TT DEL % 100% 100% 55% Ile133Thr % 100% 70% 55% G INS % 100% 100% 55% 163bp DEL codon % 100% 100% 55% T INS % 100% 100% 55% Ile133Asn % 100% 100% 55% T INS % 100% 100% 55% Ala134Val % 100% 100% 55% CC INS % 100% 100% 55% Thr135Pro % 100% 95% 55% Thr135Asn % 100% 100% 55% C INS % 100% 100% 55% A INS % 100% 100% 55% A INS % 100% 100% 55% Asp136Gly % 100% 75% 55% Asp136His % 100% 100% 55% C INS % 100% 100% 55% Asp136Tyr % 100% 100% 55% GC INS % 100% 100% 55% His137Pro % 100% 100% 55% G INS % 100% 100% 55% His137Arg % 100% 100% 55% His137Asp % 100% 100% 55% Cys138Ser % 100% 100% 55% Cys138Tyr % 100% 100% 55% Cys138STOP % 100% 100% 55% Cys138Arg % 100% 100% 55%

27 Cys138Trp % 100% 100% 55% Cys138Cys % 100% 100% 55% T DEL % 100% 100% 55% Val139Gly % 100% 94% 55% Val139Met % 100% 100% 55% Val139Ala % 100% 81% 55% Val139Leu % 100% 100% 55% G INS % 100% 100% 55% TG DEL % 100% 100% 55% GC INS % 100% 100% 55% G INS % 100% 100% 55% G DEL % 100% 100% 55% 68bp DEL % 100% 100% 55% Arg140Pro % 100% 100% 55% Arg140Gly % 100% 100% 55% Gln141Pro % 100% 100% 55% 10bp INS codon % 100% 100% 55% 13bp INS % 100% 100% 55% Gln141stop % 100% 100% 55% C INS % 100% 100% 55% Thr142Ala % 100% 100% 55% Thr142Lys % 100% 100% 55% Thr142Met % 100% 100% 55% GG INS % 100% 100% 55% Ala143Pro % 100% 100% 55% Ala143Thr % 100% 25% 55% Ala143Gly % 100% 100% 55% GAG DEL % 100% 100% 55% Ala146Thr % 100% 100% 55% Ala146Glu % 100% 100% 55% Ala146Val % 100% 100% 55% Ala146Pro % 100% 100% 55% T INS % 100% 100% 55% CG INS % 100% 100% 55% Arg148Ser % 100% 100% 55% 12bp INS % 100% 100% 55% 12bp INS % 100% 100% 55% G DEL % 100% 100% 55% 8-bpDEL % 100% 100% 55% GC INS % 100% 100% 55% DEL % 100% 100% 55% Leu151Ser % 100% 100% 55%

28 T DEL % 100% 100% 55% T INS % 100% 100% 55% Thr153Asn % 100% 100% 55% Thr153Ile % 100% 100% 55% Arg154Gly % 100% 75% 55% Val155Met % 100% 100% 55% Val155Glu % 100% 100% 55% Val155Gly % 100% 100% 55% Val155Ala % 100% 20% 55% CT INS codon % 100% 100% 55% T INS % 100% 100% 55% Leu156Pro % 100% 100% 55% Leu156Gln % 100% 100% 55% Val157Ala % 100% 100% 55% Val157Gly % 100% 50% 55% Val158Gly % 100% 100% 55% Leu159Pro % 100% 100% 55% Leu159Arg % 100% 100% 55% C INS % 100% 100% 55% Thr160Lys % 100% 100% 55% Thr160Pro % 100% 100% 55% 6bp INS codon % 100% 100% 55% Thr160Ala % 100% 50% 55% Ala161Pro % 100% 100% 55% Gly162Ser % 100% 100% 55% Gly162Asp % 100% 100% 55% GT DEL % 100% 100% 55% Gly162Val % 100% 100% 55% 11bp DEL % 100% 100% 55% 10bp DEL % 100% 100% 55% 1bp DEL % 100% 100% 55% Val163Ala % 100% 50% 55% Val163Arg % 100% 100% 55% Ser164Pro % 100% 100% 55% T DEL % 100% 100% 55% C INS % 100% 100% 55% C INS % 100% 100% 55% 1bp INS % 100% 100% 55% CG INS % 100% 100% 55% Thr167Pro % 100% 100% 55% Thr168Pro % 100% 100% 55% Thr168Asn % 100% 100% 55% 6bp DEL % 100% 100% 55%

29 Val169Ala % 100% 100% 55% C DEL % 100% 100% 55% Ala171Val % 100% 71% 55% Ala171Thr % 100% 75% 55% Ala171Pro % 100% 100% 55% GC DEL % 100% 100% 55% Ala171Glu % 100% 100% 55% Leu172Pro % 100% 96% 55% Leu172Ala % 100% 100% 55% C DEL % 100% 100% 55% CG INS % 100% 100% 55% T DEL % 100% 100% 55% G INS % 100% 100% 55% Leu172Arg % 100% 100% 55% 5-bp INS % 100% 100% 55% G INS % 100% 100% 55% Gln174STOP % 100% 100% 55% GG INS % 100% 100% 55% T INS % 100% 100% 55% Met175Val % 100% 100% 55% Met175Arg % 100% 100% 55% Met175Thr % 100% 100% 55% Thr177Pro % 100% 100% 55% 6bp DEL % 100% 100% 55% CG INS codon % 100% 100% 55% C DEL % 100% 100% 55% 6bp DEL % 100% 100% 55% Ser179Arg % 100% 100% 55% Ser179Ile % 100% 100% 55% Ser179Cys % 100% 100% 55% Val180Phe % 100% 100% 55% Val180Ala % 100% 100% 55% Val180Gly % 100% 100% 55% A DEL % 100% 100% 55% 4bp DEL % 100% 100% 55% Leu182Ser % 100% 100% 55% Cys184Tyr % 100% 100% 55% STOP187Gly % 100% 100% 55% Arg191Glu % 100% 100% 55% T DEL % 100% 100% 55% Leu4Trp/Ser65Ser % 100% 100% 55% Asp8Tyr/Leu34Arg % 100% 100% 55%

30 Gln10Arg/Ser65Ser % 100% 100% 55% Ile5Met/TG INS codon % 100% 100% 55% Gln10Pro/Val128Phe % 100% 100% 55% Thr100Ala/Arg148Cys % 100% 100% 55% Val180Ala/C DEL codon % 100% 100% 55% A-11G/Ala161Ala % 100% 100% 55% A-11G/A DEL % 100% 100% 55% A-11G/Ser65Ser % 100% 100% 55% G DEL -115/Val128Gly % 100% 100% 55% G INS -117/His137Arg % 100% 100% 55% C DEL -125/Ser65Ser/GC INS 528 C DEL - 125/Ser65Ser/Trp68Arg % 100% 100% 55% % 100% 100% 55% Val130Gly/GG INS % 100% 100% 55% Arg140Cys/T DEL % 100% 100% 55% Cys14Trp/Tyr103His % 100% 100% 55% Cys14STOP/A DEL % 100% 100% 55% Cys14Arg/Ser65Ser % 100% 100% 55% Arg140Ser/8bp DEL % 100% 100% 55% A-15C/Leu4Ser % 100% 100% 55% T DEL 43/Ser65Ser % 100% 100% 55% Ala152Gly/7bp DEL % 100% 100% 55% Thr153Ile/Asp159Glu % 100% 100% 55% Arg154Met/Thr160Ala % 100% 100% 55%

31 9bp INS 484/2bp DEL % 100% 100% 55% Asp49His/Ala102Val % 100% 100% 55% 3bp DEL 57/4bp DEL 62/5bp % 100% 100% 55% DEL 70 Gly78Ser/DEL % 100% 100% 55% Val21Glu/Gln122Leu/Arg140 Gly/Asp145His/Gly150Asp/V al155met/ala165asp/ala178 Asp/Ser179Cys/Val183Phe % 100% 100% 55% Ala3Glu/Arg140His % 100% 100% 55% AG DEL 94/GA DEL % 100% 100% 55% Tyr34Asp/Ala134Ser % 100% 100% 55% Leu35Arg/His82Asp % 100% 100% 55% Ala39Ala/His57Asp % 100% 100% 55% Ala165Thr/2bp DEL % 100% 100% 55% Thr47Ala/Arg148Cys % 100% 100% 55% His51Gln/Ala165Thr % 100% 100% 55% Ile52Ile/Leu182Phe % 100% 100% 55% Asp53Asn/5bp INS % 100% 100% 55% Asp56Cys/His57Pro/Phe58Le % 100% 100% 55% u Thr61Ser/Val139Ala % 100% 100% 55% Thr61Arg/Trp68Leu % 100% 100% 55% Ser65Ser/Gly105Asp/ A DEL % 100% 100% 55% 535 Ser65Ser/A DEL % 100% 100% 55%

32 Ser65Ser/Asp110Tyr/A DEL % 100% 100% 55% 535 Ser65Ser/Val128Gly % 100% 100% 55% Ser67Leu/GG INS % 100% 100% 55% Ser65Ser/G INS % 100% 100% 55% Ser65Ser/G INS % 100% 100% 55% Ser65Ser/Asp136Asn % 100% 100% 55% Ser65Ser/Gln141Pro % 100% 100% 55% Ser65Ser/Thr142Pro % 100% 100% 55% Ser65Ser/Leu172Pro % 100% 100% 55% Ser65Ser/GC INS % 100% 100% 55% Ser65Ser/Ser179Arg % 100% 100% 55% Ser65Ser/A DEL % 100% 100% 55% Ser65Ser/His82Asp % 100% 100% 55% Ser65Ser/2bp DEL % 100% 100% 55% Thr135Ala/Arg154Thr/ACC % 100% 100% 55% DEL 207 T-7C/Thr114Ala % 100% 100% 55% Ala79Val/T INS % 100% 100% 55% His82Arg/Val130Ala % 100% 100% 55% Lys96Thr/G DEL % 100% 100% 55% GENE DEL % 100% 100% 55% 200+bp DEL % 100% 100% 55% Note: Mutations within isolates with multiple mutations in pnca were considered together and exclusive from other isolates with the same mutations. Isolates with multiple mutations are denoted with a slash ( / ) between the different mutations.

33 Supplemental Table 7. List of Excluded Mutations Due to Incorrect Reporting of Reference Amino Acid in PZase in Mycobacterium tuberculosis clinical isolates Reference and Author PubMed ID Year Published Number of Pyrazina mide- Resistant Isolates Codon Number H37rv Amino Acid Mutation Found in the Correspon ding Study Wade et Trp Tyr118Arg al. Sachais et Asp Phe80Val al. Jonmalun Gln Thr174Ile g et al. Jonmalun Ser Gly74Arg g et al. Jonmalun Gln Gly122Ser g et al. Jnwali et al Thr Tyr114Ser Note: H37rv reference is under GenBank Accession Number NC_ Supplemental Table 8. Statistical Analysis of the Coding Region or the Coding Region and the Promoter Region of pnca in Mycobacterium tuberculosis Pyrazinamide-Resistant Isolates for Molecular-Diagnostic Tests pnca Sensitivity Specificity Negative Predictive Value Positive Predictive Value Coding Region Only 79% 90% 88% 88% Coding + Promoter Regions 83% 91% 86% 88% Supplemental Table 9. List and frequency of PZase mutations found in phenotypically Pyrazinamide-Susceptible Mycobacterium tuberculosis isolates. Number of Unique Mutation Number of Susceptible Isolates Resistant with Mutation C DEL

34 A-11C 2 2 A-11G 4 36 A-11T 1 0 C INS Met1Ile 1 5 Ile6Leu Val7Gly 1 11 Gln10Arg 1 8 Asn11Asp 1 0 Asp12Gly 1 13 Asp12Ala 3 31 Phe13Leu 1 3 Phe13Ser 1 2 Cys14Arg 1 13 Cys14Gly 3 2 Val163Ala 1 1 Gly17Ser 1 3 Val21Ala 3 1 Leu27Pro 1 7 Ala28Thr 1 0 Ile31Ser 3 4 Asp33Ala 1 1 Leu35Pro 1 8 Leu35Arg 2 1 Ala38Ala 1 0 His43Tyr 1 0 Val45Ala 1 3 Ala46Val 3 12 Thr47Ala 9 18 Lys48Glu 2 3 Asp49Glu 1 0 His51Arg 2 24 His51Pro 1 10 Pro54Gln 1 7 Pro54Leu 2 25 His57Tyr 1 7 Phe58Leu 3 13 Gly60Gly 2 0 Tyr64Ser 1 0 Ser65Ser Ser65Pro 2 1 TCG INS Trp68Arg 1 13

35 Trp68Gly 1 16 His71Arg 3 9 Thr76Pro 1 40 Phe81Ser 1 0 Phe81Val 1 2 His82Asp 1 2 Leu85Pro 1 41 Phe94Cys 1 5 Phe94Leu 1 14 Lys96Glu 1 13 Gly97Asp 1 13 Thr100Ile 1 0 Ala102Arg 1 0 Ala102Pro 1 0 Ala102Val 4 5 Tyr103STOP 1 19 Tyr103Cys 1 3 Trp119Cys 1 2 Leu119Pro 1 0 Gly124Asp 1 0 Val128Gly 1 15 Val128Phe 1 0 Val130Ala 1 4 Ile133Thr 7 16 Thr135Ser 1 0 Thr135Pro 1 21 Asp136Gly 2 6 His137Cys 2 0 Val139Ala 5 21 Val139Gly 1 16 Arg140His 2 0 Ala143Thr 3 1 Ala143Val 1 0 Arg154Gly 2 6 Arg154Ser 3 0 Val155Ala 4 1 Val155Leu 1 0 Val157Gly 1 1 Leu159Val 1 0 Thr160Ala 1 1 Thr168Ile 2 0 Ala171Val 2 5 Ala171Thr 1 3

36 Leu172Pro 1 22 Met175Ile 2 11 Leu182Trp 1 0 STOP187Ala 1 0 Stop187Arg 2 0 Trp119stop/Thr168Ile 1 0 C DEL -124/Ser65Ser 1 0 Arg140His/Gly150Gly/Ala165Thr 1 0 Arg140His/Ala165Thr 2 0 Supplemental Table 10. Percentages of Pyrazinamide Resistant Mycobacterium tuberculosis with or without Detectable Pyrazinamidase Activity or PZase Mutations Resistant isolates with PZase activity and at least one mutation in PZase Resistant isolates with PZase activity and no mutation in PZase Resistant isolates with no PZase activity and no mutations in PZase 6%* 6%* 3%* *Percentage of 1979 PZA-resistant isolates that underwent enzymatic testing Supplemental Table 11. List of Mutations in PZase or the Promoter Region Found in Pyrazinamide-Resistant M. tuberculosis Isolates that Tested Positive or Negative for Pyrazinamidase Activity. PZA-Resistant Associated Mutations in Pzase Number of PZA- Resistant Isolates with Positive Pzase Activity Number of PZA-Resistant Isolates with Negative Pzase Activity T-10C 1 0 Ala3Ser 1 0 Asp12Gly 1 8 Asp12Asn 0 4 Asp12Ala 1 15 Gly23Ala 1 0 Gly24Asp 1 1 Ala28Val 1 0 lle31thr 4 0 Tyr34Asp 1 9 Leu35Pro 1 2 Leu35Arg 1 0

37 His43Pro 1 1 Lys48Thr 2 0 Asp49Asn 1 1 Pro54Leu 1 10 His57Pro 1 6 Pro62Arg 1 1 Pro62Gln 1 2 Pro62Leu 1 5 Asp63Ala 1 0 Ser65Pro 1 0 His71Thr 1 1 Thr76Pro 2 15 Thr76Ile 1 1 Gly78Cys 1 0 Phe81Val 1 1 His82Arg 1 0 Pro83Arg 1 0 Phe94Leu 1 0 Ala102Val 1 3 Asn112Tyr 1 2 Leu116Pro 1 2 G INS Gly132Asp 1 2 Ile133Thr 1 3 A INS Val139Ala 1 3 Ala143Gly 1 0 Leu151Ser 1 4 Thr160Ala 1 0 Gly162Ser 1 0 Ala171Val 1 1 Met175Val 1 1 Thr177Pro 2 0 Ser65Ser/Leu172Pro 1 0

genotypic, and enzymatic tests. Based on the three tests eight groups have been defined.

38 Supplemental Figure 2. Observed stratification of clinical isolates included in this study and the conclusion (typical diagnostic decision) based on three phenotypic, genotypic, and enzymatic tests. Based on the three tests eight groups have been defined. Groups 2 and 7 include isolates that produced concordant results in all three tests. The remaining six groups contain isolates with the results of one test to be inconsistent with the other two. Notation: DST=Drug Susceptibility Testing; WT=Wild Type; Molecular Test (any sequencing technology, e.g. whole genome sequencing, Sanger, or other PCR based methods)

Nomenclature What is in a name? My name Joseˊ Jimenez = Bill Dana John L.C. Savony = Frank Fontaine

Nomenclature What is in a name? My name Joseˊ Jimenez = Bill Dana John L.C. Savony = Frank Fontaine Three categories of Amyloid Terms 1. Amyloidologists, Amyloid Centers, Researchers official nomenclature