Title: Use of mycobacteriophage qpcr on MGIT broths for a rapid tuberculosis antibiogram

Transcription

1 JCM Accepts, published online ahead of print on 26 February 2014 J. Clin. Microbiol. doi: /jcm Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 Title: Use of mycobacteriophage qpcr on MGIT broths for a rapid tuberculosis antibiogram 2 3 Running title: phage qpcr for Tb antibiogram Authors: Suporn Foongladda 1, Wiphat Klayut 1#, Rattapha Chinli 1#, Suporn Pholwat 2, Eric R. Houpt 2* Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand 1 Division of Infectious Diseases and International Health, University of Virginia * Corresponding author. Mailing address: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA Erh6k@virginia.edu # These authors contributed equally to this work 16

2 Abstract Phenotypic culture-based drug susceptibility testing for Mycobacterium tuberculosis (TB) is valuable to identify 4-6 active drugs for individualized MDR TB regimens. Current culturebased methods are slow, however, therefore we evaluated a rapid mycobacteriophage based qpcr assay for use directly on M. tuberculosis-positive MGIT broths. We compared phage qpcr, using a simple cutoff of 3 for Cq (Cq starting phage Cq Tb plus drug), on 325 clinical M. tuberculosis MGIT broth cultures versus the respective subcultured isolate tested by agar proportion. The median accuracy for the 13 drugs/concentrations tested was 98%, with most discrepancies being false resistant results. Performance on greater numbers of resistant strains on 393 isolates grown on Löwenstein-Jensen media, showed similar findings, with a median accuracy, sensitivity, and specificity of 97%, 90%, and 99%, respectively. This rapid culturebased DST methodology can be performed for any drug on TB positive MGIT broths, with a specimen to antibiogram turnaround time of approximately 23.9 days, compared with waiting 58.6 days for isolate growth on solid media followed by agar proportion DST.

3 Introduction Drug susceptibility testing (DST) for Mycobacterium tuberculosis is important for individualized care, particularly in the setting of multidrug resistant (MDR) TB. The value of DST on improved MDR TB outcomes is supported by meta-analyses that show, for instance, that the odds of treatment success are two-fold higher if initially treated with 4 or more active drugs defined on the basis of DST (2). Higher levels of resistance beyond MDR and XDR are further associated with poor outcome (4). However methods for TB DST are laborious and limited. Molecular DST offers a great advance in terms of speed and in identifying MDR (e.g., Cepheid Xpert MTB/RIF) or XDR (e.g., Hain MTBSLDplus line probe assay), however identifying MDR or XDR does not equate with identifying 4 or more active drugs. Molecular results generally yield only drug class information, while MDR clinicians must make within-injectable or withinfluoroquinolone drug choices, and we use drugs such as ethionamide, p-aminosalicylic acid, cycloserine, clofazimine, linezolid, bedaquiline, or other drugs to fill out a regimen, yet molecular assays for these drugs are currently nonexistent. Furthermore, MDR treatment regimens generally exceed 18 months and patients are frequently not able to tolerate a single regimen due to side effects, which means drug changes may be necessary later in the regimen. For this reason, while we share great enthusiasm for molecular methods, in our view culturebased phenotypic methods that can ascertain susceptibility to any and all anti-tb drugs will continue to be necessary for optimal individualized care for the foreseeable future. To this end, faster culture-based methods are needed, since typical methods are slow, requiring readings at 4 to 6 weeks, which delays the detection of drug resistance and risks inappropriate treatment early at the time of peak burden and transmissibility. This has been our rationale for the development and evaluation of a rapid mycobacteriophage quantitative PCR

4 based method. In recent work we have described the methodology and its performance on 32 clinical isolates in our laboratory (8), and examined its speed and concordance versus the proportion method using Löwenstein-Jensen media, MGIT 960 SIRE AST, Xpert MTB/RIF, GenoType MTBDRplus line probe assay, and MYCOTB MIC plate on 87 mostly MDR isolates in Bangladesh (3). In the present work we evaluate the method in a high volume clinical mycobacteriology laboratory against the gold-standard 7H10 agar proportion method and describe its use directly on primary MGIT broths, usually the first positive culture source, which can obviate the need for subculture and further decrease turnaround time MATERIALS AND METHODS Mycobacterial strains and culture conditions. Mycobacterial strains used in this study included 325 M. tuberculosis positive MGIT broths and 456 clinical M. tuberculosis isolates grown on solid Lowenstein-Jensen (LJ) media. Additionally the following reference strains were used: M. tuberculosis H37Rv (ATCC 27294), M. smegmatis (ATCC 607), 7 M. abscessus, 1 M. chelonae complex, 7 M. fortuitum complex, 4 M. kansasii, 3 M. simiae, 2 M. gordonae, 4 M. scrofulaceum, 6 M. avium, 7 M. avium complex, and 5 M. intracellulare. M. tuberculosis was confirmed by using a laboratory-developed sequence specific FRET probe (5) while nontuberculous mycobacteria (NTM) were identified by INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium). MGIT broth was subcultured on to LJ media then incubated at 37 o C for 3 weeks to obtain isolates for agar proportion comparison. All clinical isolates were obtained from the Mycobacteriology Service Unit, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. All work was approved by

5 the Siriraj Institutional Review Board of the Human Research Protection Unit of Medicine, Mahidol University, and UVA Human Investigation Committees. Antimicrobial agents and standard agar proportion method. Drugs used and standard antimicrobial susceptibility tests were described previously (8) except the purified powder of moxifloxacin was used instead of water injection form in this study. Optimization of D29 phage assay conditions. The D29 phage used in this study were propagated and quantified as described previously (8). Phage stability was determined by storing D29 phage at -20 C, 4 C, 25 C, and 37 C, then quantifying the number of phage weekly for 12 weeks and at weeks 24 and 48. The capability of D29 phage to infect non-tuberculous mycobacteria (NTM) was examined on 46 clinical NTM isolates, M. smegmatis ATCC 607, and M. tuberculosis H37Rv (control). Colonies on solid LJ media were suspended and adjusted to 0.5 McFarland, diluted, and inoculated into M7H9 plus 10% OADC and 1 mm CaCl 2, and incubated 48 h at 37 C. Then 10 μl of ~ PFU/ml of D29 phage was added and further incubated for 48 h, followed by real-time PCR for D29 phage DNA. Evaluation of D29 phage assay using primary growth from MGIT broth as inoculum. 325 M. tuberculosis positive MGIT broths were tested for drug susceptibility using the D29 phage assay on 11 drugs (isoniazid (INH), rifampin (RIF), ethambutol (EMB), streptomycin (STR), amikacin (AMK), kanamycin (KAN), capreomycin (CAP), ofloxacin (OFX), moxifloxacin (MXF), ethionamide (ETH), and p-aminosalicylic acid (PAS)) using where possible recommended critical concentrations for MGIT 960 broth media (1, 7). Inocula were prepared by centrifugation of a primary MGIT clinical isolate tube at 3500 rpm for 10 min then 1 ml of sediment was collected, transferred, centrifuged at rpm for 10 min and the pellet was thoroughly suspended in ~250 μl of M7H9 plus 10% OADC and 1 mm CaCl 2, allowed to

6 settle for min, then transferred to a new tube and used as inoculum. Ten μl was inoculated into each well of a 96 well plate containing 90 μl of drug-containing media and incubated at 37 o C for 48 h. Then D29 phage was added for 48h as above followed by real-time PCR (PCR reagents were sealed inside a biosafety cabinet). M. tuberculosis H37Rv was used as a quality control for each batch of drug. Evaluation of D29 phage assay using LJ isolates. 456 isolates of M. tuberculosis were tested for drug susceptibility using the D29 phage assay. Tb suspensions (0.5 McFarland) were diluted 1:10 in M7H9 plus 10% OADC and 1 mm CaCl 2 to yield inoculum ~1x10 6 cfu/ml, 10 µl was used as inoculum as described above. D29 phage qpcr assay. The primers/probe, real-time PCR platform, and PCR cycling condition as described previously (3) were minor modifications, including a PCR total volume 20 µl, and we used 2X itaq Universal Probes Supermix (Bio-Rad, Hercules, CA). The PCR quantification cycle (Cq) of D29 phage alone (starting phage), of TB isolates in drug-free medium followed by phage (control TB), and TB isolates in drug-containing medium followed by phage were analyzed. Statistical analysis. Means were compared using t test or medians using Mann-Whitney test, as appropriate. All P values were two-tailed. Data shown as mean ± standard deviation. RESULTS Phage assay optimization. Phage stability was tested at -20 C, 4 C, 25 C and 37 C, with the pfu/ml of phage quantified weekly for 12 weeks and at week 24 and 48. This revealed that storage at 4 C provided consistent quantities of viable phage up to 8 weeks with only slight decrement at week 48. By contrast, storage at 25 o C and -20 o C led to loss of phage viability in 5-8 weeks (Supplemental Figure 1A). The capability of D29 phage to infect non-tuberculous

7 mycobacteria (NTM) was important to determine given our intended use of testing primary MGIT broths. We evaluated 46 clinical NTM isolates and M. smegmatis ATCC CFU of each strain was cultured at 37 C for 48 h prior to adding 10 2 PFU of phage for 48h, followed by qpcr. The Cq (Cq phage alone Cq NTM plus phage) were calculated as an indication of how much phage replication could have occurred (Supplemental Fig. 1B,C). A minority of M. abscessus, M. chelonae complex, and M. fortuitum complex (and of course M. smegmatis, as is known) revealed phage amplification. Namely, Cq were not statistically significant different from control M. tuberculosis H37Rv. However, D29 phage did not amplify in any of our clinical slow growing NTM. Thus, caution should be exercised when using the phage assay in rare cases of mixed rapid grower/tb specimens. Interpretation criteria. The hypothesis of the assay is that D29 mycobacteriophage will infect and increase its DNA in viable Tb cells, and that this DNA can be quantified by real-time PCR to discern Tb viability/resistance amidst drug treatment. Across a wide dynamic range, a change of 3 qpcr quantification cycles (Cq) corresponded to approximately a 10 change in D29 plaque-forming units (Supplemental Figure 1D). Additionally, of the 26 independent experiments performed in this study, the average Cq of starting phage was 30.8 ± 1.5, thus 2 standard deviations = 3 Cq. For these reasons, for any isolate, we required that the ΔCq of control TB (Cq starting phage Cq MGIT broth/isolate) equal or exceed 3.0 in order to be certain (a) that the Tb was viable enough to allow at least 10-fold phage replication and (b) that its Cq was reliably dissimilar from starting phage. If these two criteria were not met the assay was deemed invalid for the specimen. When a valid result was obtained, there are several metrics that can be used to determine resistance or susceptibility: one can use (Cq Tb control Cq Tb + drug) which we used in the

8 original manuscript (8) and reflects how susceptible the TB is to the drug relative to no drug at all; a ratio (Cq starting phage Cq Tb plus drug) / (Cq starting phage Cq Tb control) which we used in our subsequent manuscript (3) and reflects the relative amount of growth in drug versus control, akin to the agar proportion method. Similarly, one can use (Cq starting phage Cq Tb + drug) which reflects how resistant the Tb is to a drug, whereby, for the same reasons cited above, < 3.0 can be used as a cutoff for susceptible while 3.0 can be considered resistant in that there is some statistically significant phage replication. We evaluated these 3 different metrics (Cq Tb control Cq Tb + drug)(8); (Cq starting phage Cq Tb + drug) / (Cq starting phage Cq Tb control) (3); (Cq starting phage Cq Tb + drug) and found them to perform similarly versus agar proportion. Specifically the median sensitivities were 75, 89, 90%, and specificities 97, 98, 99%, respectively, for the eleven drugs tested, P = NS. For this reason, for our laboratory we chose the universal (Cq starting phage Cq Tb + drug) ΔCq cutoff of 3.0 because it was easy to compute and understand = how resistant the TB is in the setting of drug. Evaluation of D29 phage assay using primary MGIT broths as inoculum. A total of 325 clinical M. tuberculosis MGIT broth cultures were assayed by phage qpcr and compared with their respective subcultured isolate tested by agar proportion. Sixty-one of 325 (18.5%) broths could not be compared because a pure Tb isolate could not be subcultured for agar proportion (n=22), insufficient growth during agar proportion testing (n=10), and/or no phage replication (n=31). All MGIT broths were tested for first line drugs (INH, RIF, and EMB) and any resistant isolates (n=113) were additionally tested for second line drugs. The median accuracy for the 13 drugs/concentrations tested was 98%, and ranged from 76% (for PAS) to 100% (Table 1). Taking the agar proportion result as gold standard, most discrepant phage results were false resistant (n=37) as opposed to false susceptible (n=21) and the vast majority

9 (49/58=85%) were from ETH and PAS. The scatter plots of phage qpcr ΔCq versus agar proportion is shown in Figure 1 and shows how the simple cutoff of 3 performs for INH, RIF, EMB, STR, CAP, MFX, ETH and PAS (for other drugs/concentrations see Supplemental Figure 2). Evaluation of D29 phage assay using isolates. While this confirmed that the assay worked well on MGIT broths, most specimens were drug susceptible. Thus we selected an additional 456 previously collected clinical isolates from our laboratory with known resistance to one or more drugs. These isolates were grown on LJ media then tested by phage qpcr and retested with agar proportion on 7H10 media. Sixty-three yielded no phage growth, leaving 393 for analysis, all of which were tested for INH, RIF, and EMB, and any resistant isolates (n=233) further tested for 2 nd line drugs. Overall accuracy ranged from 86% (for ETH) up to 100% with a median accuracy of 97% for the 13 drugs/concentrations tested (Table 2). Overall general the sensitivity for detecting resistance was good (median 90% for all 13 drugs/concentrations tested) yet modest for detecting EMB-resistance (59-85% for low or high dose EMB in agar proportion). On the other hand the specificity for detecting susceptibility was excellent (median 99%). As with the MGIT broths, most discrepant phage results were false resistant (n=100) as opposed to false susceptible (n=46) and 34% (50/146) were from ETH and PAS. Of the false resistant results, most had a lower ΔCq than the true resistant results (see parentheses, Table 2), suggesting a lower level of resistance, however these ΔCq differences were only statistically significant for OFX, MXF, and ETH. This can be seen in the scatter plots of phage qpcr and agar proportion (Supplemental Figure 3), which suggests shifting the cutoff from 3 upwards to 5-10 would improve accuracy for OFX and MFX, however power remained limited given low numbers of resistant isolates for quinolones.

10 Turnaround time of the assay. The average time from specimen to growth in MGIT broth was 14.1 ± 8.9 days (n = 325) versus 31.8 ± days for LJ isolates (time data collected on only n = 177). Thereafter species identification was performed in batches and thus required 5.8 ± 3.6 days. For susceptibility testing the duration of phage qpcr assay was always 4 days while agar proportion required at least 21 days. Therefore the average turnaround time from specimen to DST was 23.9 days for MGIT broths/phage qpcr DST, 41.6 days for isolates on LJ/phage qpcr DST, 54.9 days for MGIT broths/agar proportion DST (adding a typical 2 weeks to subculture the MGIT broths), and 58.6 days for isolates on LJ/agar proportion DST DISCUSSION The main finding of this work is the demonstration of a rapid culture-based DST methodology that can be performed for any Tb drug on MGIT broths. The assay offered very good accuracy for the 13 drugs/drug concentrations tested, compared to waiting for the respective subcultured isolate and agar proportion results. The method allows for a full antibiogram for cultured TB, typically within 24 days, which is about 35 days faster than waiting for LJ growth and performing agar proportion. This is a time of great uncertainty with potential for inappropriate therapy and ongoing transmission. We also used a simple metric (Cq starting phage Cq Tb plus drug) that was easy to operationalize. In our large Thai hospital laboratory we are making the qpcr method a special request for clinicians that want rapid results for several drugs such as in the MDR or MDR-suspected scenario. In this latter setting the clinician wants confidence in identifying an MDR regimen early, with at least 4 active drugs, while awaiting agar proportion results to finalize and then adjust the regimen. The typical first step is for the liquid media to turn positive, and the

11 specificity of the mycobacteriophage for TB allows testing of the broth immediately, without the need for subculture to obtain a pure isolate. Later, if desired, the isolate, either subcultured from the MGIT broth or from the solid media inoculated from the sputum, could be tested with conventional culture-based DST (e.g., MGIT or TREK or agar proportion), obtaining a confirmatory result weeks thereafter. Using several such methods sequentially is commonplace in well-resourced laboratory settings and this staged approach highly acceptable to clinicians. There were limitations to this work. First we noticed that a minority of rapid grower mycobacteria supported phage replication, thus the rare MGIT broth that was a mixed TB/rapid grower NTM could lead to spurious results (however one should notice that the MGIT broth looks contaminated anyway). Furthermore we found that approximately 10% of the tuberculosis-positive MGIT broths did not sufficiently support phage replication, thus the assay will not work on all isolates, presumably due to properties intrinsic to certain bacilli. Importantly however this failure rate is similar to the 10% of MGIT broths that failed to yield an agar proportion result because of insufficient growth or inability to obtain a pure culture. Power was limited to measure the sensitivity of detecting resistance, even though this is a very high volume laboratory (>1500 Tb isolates per year) in the largest hospital in Thailand. Performance was excellent on the prospective primary MGIT culture broths but sensitivity decreased slightly on the banked resistant isolates, particularly for Ethambutol (59%). We and others have found ethambutol to be a particularly problematic drug for DST (3, 6), so part of this low sensitivity could reflect problems with the gold standard. However this 59% sensitivity still retained a positive predictive value of 89% and a negative predictive value of 97%, and for all other drugs the negative predictive values were even higher (Table 1). This NPV means that if included EMB in a regimen on the basis of phage DST they would have a 97% likelihood that

12 the agar proportion test would ultimately result susceptible. Thus the phage DST assay, at least on this study population, would be highly useful as an early indicator of 4-6 active drugs. PPV were at times lower, which means ones confidence in withholding drugs on the basis of a resistant phage qpcr result would be less, particularly for ETH and PAS. However this scenario is still clinically acceptable the clinician could easily defer PAS/ETH on this basis and revisit the regimen when the agar proportion results are final. As for why the PAS and ETH results were particularly discrepant, we suspect this is because these drugs are not very active, or not very active within a 48 hour time frame, such that even susceptible isolates still allowed significant early phage replication. In summary, we found the phage qpcr method to be a robust tool for early DST on MGIT broths. It can yield a complete antibiogram, and the performance characteristics in this setting exhibited a high NPV, such that it can be used to safely construct an MDR regimen of 4-6 active drugs while awaiting final confirmatory culture-based DST ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant R01 AI (to E.H.). We thank Jean Gratz for critical review of the manuscript and Suzanne Stroup for assisting with procuring reagents. 257

13 References Policy guidance on drug-susceptibility testing (DST) of second-line antituberculosis drugs, vol. WHO/HTM/TB/2008. World Health Organization. 2. Ahuja, S. D., D. Ashkin, M. Avendano, R. Banerjee, M. Bauer, J. N. Bayona, M. C. Becerra, A. Benedetti, M. Burgos, R. Centis, E. D. Chan, C. Y. Chiang, H. Cox, L. D'Ambrosio, K. DeRiemer, N. H. Dung, D. Enarson, D. Falzon, K. Flanagan, J. Flood, M. L. Garcia-Garcia, N. Gandhi, R. M. Granich, M. G. Hollm-Delgado, T. H. Holtz, M. D. Iseman, L. G. Jarlsberg, S. Keshavjee, H. R. Kim, W. J. Koh, J. Lancaster, C. Lange, W. C. de Lange, V. Leimane, C. C. Leung, J. Li, D. Menzies, G. B. Migliori, S. P. Mishustin, C. D. Mitnick, M. Narita, P. O'Riordan, M. Pai, D. Palmero, S. K. Park, G. Pasvol, J. Pena, C. Perez-Guzman, M. I. Quelapio, A. Ponce-de-Leon, V. Riekstina, J. Robert, S. Royce, H. S. Schaaf, K. J. Seung, L. Shah, T. S. Shim, S. S. Shin, Y. Shiraishi, J. Sifuentes-Osornio, G. Sotgiu, M. J. Strand, P. Tabarsi, T. E. Tupasi, R. van Altena, M. Van der Walt, T. S. Van der Werf, M. H. Vargas, P. Viiklepp, J. Westenhouse, W. W. Yew, and J. J. Yim Multidrug resistant pulmonary tuberculosis treatment regimens and patient outcomes: an individual patient data meta-analysis of 9,153 patients. PLoS Med 9:e Banu, S., S. M. Rahman, M. S. Khan, S. S. Ferdous, S. Ahmed, J. Gratz, S. Stroup, S. Pholwat, S. K. Heysell, and E. R. Houpt Discordance across several drug susceptibility methods for drug-resistant tuberculosis in a single laboratory. J Clin Microbiol. 4. Falzon, D., N. Gandhi, G. B. Migliori, G. Sotgiu, H. S. Cox, T. H. Holtz, M. G. Hollm-Delgado, S. Keshavjee, K. DeRiemer, R. Centis, L. D'Ambrosio, C. G. Lange, M. Bauer, and D. Menzies Resistance to fluoroquinolones and second-line injectable drugs: impact on multidrug-resistant TB outcomes. Eur Respir J 42: Foongladda, S., S. Pholwat, B. Eampokalap, P. Kiratisin, and R. Sutthent Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth. The Journal of molecular diagnostics : JMD 11: Kruuner, A., M. D. Yates, and F. A. Drobniewski Evaluation of MGIT 960- based antimicrobial testing and determination of critical concentrations of first- and second-line antimicrobial drugs with drug-resistant clinical strains of Mycobacterium tuberculosis. J Clin Microbiol 44: NCCLS/CLSI Susceptibility testing of Mycobacteria, Nocardiae, and other aerobic Actinomycetes; approved standard, vol Pholwat, S., B. Ehdaie, S. Foongladda, K. Kelly, and E. Houpt Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J Clin Microbiol 50:

14 Table 1. Performance of D29 phage qpcr assay using primary MGIT broths compared with agar proportion on the respective isolate. ND = cannot be determined. Drugs concentration (Phage/Agar) μg/ml INH (0.1/0.2) INH (0.4/1.0) RIF (1.0/1.0) EMB (5.0/5.0) EMB (7.5/10) STR (1.0/2.0) AMK (1.0/4.0) KAN (2.5/5.0) CAP (2.5/10.0) OFX (2.0/2.0) MXF (0.5/0.5) ETH (5.0/5.0) PAS (2.0/2.0) D29 phageqpcr Agar proportion Accuracy (%) Sensitivity (%) Specificity (%) NPV (%) PPV (%) ND ND ND

15 Table 2. Performance of D29 phage qpcr assay on isolates compared with agar proportion. Parentheses show average ΔCq (Cq starting phage Cq TB + drug) values for the respective isolates. *, P < 0.05 comparing false resistant and true resistant ΔCq. Drugs concentration (Phage/Agar) μg/ml INH (0.1/0.2) D29 phageqpcr Agar proportion 299 (-0.6±1.8) 4 (9.3±6.7) 8 (-1.2±2.9) 82 (13.4±4.5) Accurac y (%) Sensitivity (%) Specificity (%) NPV (%) PPV (%) INH (0.4/1.0) 176 (-0.9±2.2) 3 (12.6±6.6) 9 (-2.5±4.9) 45 (14.4±5.0) RIF (1.0/1.0) 353 (-0.6±1.8) 4 (10.2±4.1) 3 (2.5±0.4) 33 (12.6±5.7) EMB (5.0/5.0) 362 (-0.6±1.9) 2 (5.5±2.4) 12 (-0.3±1.8) 17 (11.1±5.8) EMB (7.5/10) 217 (-1.0±2.5) 3 (9.2±6.9) 2 (-3.5±0.1) 11 (9.1±5.7) STR (1.0/2.0) 306 (-0.8±2.3) 13 (10.3±4.9) 2 (-1.0±3.0) 53 (12.0±3.7) AMK (1.0/4.0) 227 (-0.7±2.6) 2 (3.5±0.2) 2 (1.5±0.2) 1 (14.4) KAN (2.5/5.0) 231 (-0.4±2.3) (14.2)

16 CAP (2.5/10.0) 228 (-0.5±2.4) 3 (6.6±5.8) 1 (1.7) 0 98 NA OFX (2.0/2.0) 218 (-0.5±2.5) 6 (3.8±0.8)* 3 (-1.5±3.5) 5 (15.6±1.9) MXF (0.5/0.5) 189 (-0.2±2.4) 3 (8.7±2.5)* 1 (-4.4) 8 (15.3±2.1) ETH (5.0/5.0) 169 (-0.4±2.6) 31 (8.8±3.6)* 2 (1.0±0.1) 30 (10.9±3.8) PAS (2.0/2.0) 194 (-0.4±2.6) 26 (7.4±3.3) 1 (2.4) 11 (9.8±3.9)

17 Figure 1. Correlation between D29 phage qpcr on primary MGIT broths versus agar proportion method on isolates. Two hundred sixty four M. tuberculosis MGIT broths were tested by phage qpcr and their respective isolate tested by agar proportion for susceptibility to the drugs and concentrations indicated. For the phage qpcr assay Tb were pre-treated with drugs or control media for 48h, followed by adding phage and incubation for 48h. Results shown are Cq (Cq starting phage Cq TB+drug ) where < 3.0 (blue line) is defined as susceptible and 3.0 is defined as resistant. Red symbols -, indicate an agar proportion result that was discrepant with D29 phage qpcr.

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