1 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Transcription

1 JCM Accepts, published online ahead of print on 18 November 2009 J. Clin. Microbiol. doi: /jcm Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Sputum Mycobacterium tuberculosis mrna as a marker of bacteriologic clearance in response to anti-tuberculosis therapy L. Li, 1* C. S. Mahan, 2,3* M. Palaci, 4 L. Horter, 3 L. Loeffelholz, 5 J. L. Johnson, 3 R. Dietze, 4 S. M. Debanne, 6 M. L. Joloba, 7,8 A. Okwera, 9 W. H. Boom, 3 and K. D. Eisenach 1 1 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 2 Department of Medicine, MetroHealth Medical Center, Cleveland, Ohio 3 Tuberculosis Research Unit, Department of Medicine, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, Ohio 4 Núcleo de Doenças Infecciosas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, Brazil 5 Normandale Community College, Bloomington, Minnesota 6 Department of Epidemiology and Biostatistics, Case Western Reserve University School of Medicine, Cleveland, Ohio 7 Joint Clinical Research Centre, Kampala, Uganda 8 Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda 9 National TB and Leprosy Control Programme, Kampala, Uganda Correspondence and requests for reprints should be addressed to Kathleen D. Eisenach, University of Arkansas for Medical Sciences, Department of Pathology, slot 517, 4301 W. Markham, Little Rock, AR eisenachkathleend@uams.edu Fax #: Running title: Sputum mrna and response to TB treatment *These authors shared first co-authorship as both contributed equally to this work.

2 Abstract Messenger RNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mrna in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mrna levels were measured in sputum specimens from patients with TB receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an IL-2 trial. In the early bactericidal activity study, sputum for quantitative culture and mrna analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, BACTEC 460 liquid culture, and mrna analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-pcr assays for several gene targets. Messenger RNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mrna correlated highly with colony forming units in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mrna was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mrna correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mrna is a reliable marker of M. tuberculosis viability. Key words: tuberculosis, RT-PCR, mrna, surrogate marker, anti-tuberculosis drugs Introduction The development of new drugs for tuberculosis (TB) treatment has been hampered by the lack of an early surrogate marker that reflects non-relapsing cure. Two month sputum culture conversion on solid medium is the best established predictor of treatment outcome (12). Early bactericidal activity (EBA), measured as the decline in sputum Mycobacterium tuberculosis colony counts (CFU) during treatment, is a commonly used tool for comparing new drugs to current drugs and

3 dose finding, but quantitative culture is time consuming and labor intensive (4). An ideal marker would measure events early during treatment and be accurate regardless of the mechanism of drug action or the regimen being tested. In addition to serial sputum culture conversion, less well-studied biomarkers of response to therapy include sputum 85B protein and mrna (17, 2), sputum cytokines (15), and whole blood bactericidal activity (18). None has been adequately validated to demonstrate long-term efficacy in Phase 3 trials. In prior work we demonstrated that levels of M. tuberculosis fbpb mrna (encoding fibronectin-binding protein, antigen 85B) and hspx (encoding alpha crystalline homologue protein) mrna decline rapidly in conjunction with M. tuberculosis colony counts after initiation of a rifampin-based standard drug therapy (2) and in a 14-day early bactericidal activity (EBA) study of rifalazil where both isoniazid and a rifamycin were given simultaneously (unpublished data). In most cases the solid medium culture was still positive when the M. tuberculosis mrna was undetectable. To further explore the application of quantitative reverse transcription (RT) PCR for detecting viable M. tuberculosis in sputum, we developed assays for additional targets in anticipation of identifying genes expressed at higher levels than fbpb and hspx mrna, so that a higher number of mrna molecules per organism would be available for detection over longer periods, i.e., weeks or months vs. days. Using newly developed assays for icl (encoding isocitrate lyase) and rrna-p1 (noncoding ribosomal promoter region) we evaluated mrna as a marker of bactericidal and sterilizing activity in sputum specimens from TB patients receiving monotherapy in an EBA study (i.e., in the absence of the RNA-polymerase inhibitor rifampin). In addition, we evaluated the effect of rifampin on the detection of mrna in patients receiving standard shortcourse chemotherapy in a trial of adjunctive IL-2 in TB treatment. Materials and Methods Patients and specimens. (i) From EBA study. As previously described, 40 Brazilian adults with smear-positive fully drug susceptible TB were randomly assigned to receive 7 days of daily

4 isoniazid (INH) 300 mg, levofloxacin 1000 mg, gatifloxacin 400 mg, or moxifloxacin 400 mg (9). Two pooled sputum samples were collected, each over a 12 h time period in the 2 days prior to treatment (baseline) and then daily for 7 days. Serial dilutions of sputum were cultured on Middlebrook 7H10 agar to obtain number of CFU/ml per sample as previously described (3). Onehalf ml aliquots of N-acetyl-L-cysteine (NALC) digested sputum were frozen at -70 C until RNA extraction. (ii) From IL-2 study. One hundred and ten Ugandan adults with smear-positive TB were randomly assigned to receive standard chemotherapy plus intradermal IL-2 or placebo (9). Sputum was collected at baseline and frequently throughout treatment for culture in BACTEC 460 liquid media and quantitative culture on Middlebrook 7H10 agar plates. Onehalf ml aliquots of NALC-digested sputum were stored at -70 C until RNA extraction. For the current sub-study, specimens from 20 randomly selected participants were chosen for mrna analyses. This included 76 sputum specimens comprised of 38 specimens from 19 subjects, 2 each at month 1; and 38 specimens from 20 participants (18 persons with 2 specimens each, and 2 persons with 1 specimen) at month 2. The studies were approved by the Ugandan National AIDS Research Subcommittee and the institutional review boards of the Universidade Federal do Espírito Santo, University of Arkansas for Medical Sciences, and Case Western Reserve University. All subjects gave informed consent. Reverse Transcription (RT)-PCR assays. RNA was isolated from sputum by organic extraction coupled with mechanical disruption as previously described (2). Using a gene-specific primer for each target (Table 1), RNA was reverse transcribed. Efficiency of RT was determined by using in vitro RNA transcripts of cloned M. tuberculosis genes. Dilutions of control transcripts ranging from 10 2 to10 5 molecules/µl in 10 ng/ yeast carrier RNA were included in each assay. The efficiency of RT was calculated as the ratio of the number of observed to expected molecules per reaction.

5 The amount of cdna was quantified by PCR with the corresponding TaqMan probe using the ABI 7900HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). PCR conditions were identical for all assays. The 20 µl reaction mixture consisted of 2 x Taqman Universal PCR master mix (ABI), 0.9 µm of each primer, and 0.25 µm of probe (Table 1). The quantity of specific target DNA was determined from the Ct value with reference to a standard curve of genomic DNA (5 to 78,125 copies) obtained from M. bovis ATCC The graphs of starting DNA concentration versus Ct value were consistently linear (R 2 = 0.99) over the range of 5 to 78,125 copies. Ct values for samples and standards fell between 21 and 38 cycles of amplification. The lower limit of detection for each of the four assays was 5 copies of cdna. RT reactions were done twice; the mean was used in calculations. The number of molecules of mrna/ml sputum was calculated by correcting for the efficiency of RT and the dilution factors involved in RT and PCR amplification, and initial sputum processing. Statistical analyses. Individual slopes and intercepts were obtained by fitting linear regressions for each patient using SAS (version 9.13; SAS, Cary, NC). Correlation coefficients for pairwise slopes and intercepts of CFU against each mrna biomarker were calculated. The rate of fall of icl mrna from day 0 to day 7 for patients in the INH arm versus the combined fluoroquinolone arms was compared using analysis of variance with correction for multiple comparisons. The Spearman rank correlation coefficient was used to assess the relationships between liquid and solid cultures, icl mrna and liquid cultures, and icl mrna and solid cultures. Results Evaluation of Mtb mrna levels during 7-day INH monotherapy. Levels of mrna for icl, rrna-p1, hspx, and fbpb in sputum specimens were measured in parallel with sputum colony counts at baseline and daily while patients were receiving INH monotherapy. The mean concentration of M. tuberculosis was / CFU/ml of sputum at baseline for all groups

6 combined with no significant difference among groups. Sputum colony counts were consistent with previous studies of this patient population (3). Likewise, levels of hspx and fbpb mrna measured in baseline sputa were consistent with our earlier findings. The mean baseline numbers of mrna/ml of sputum were 9.23 log10 for icl, 7.24 log10 for rrna-p1, 5.66 log10 for hspx, and 5.52 log10 for fbpb (Table 2). Serial measurements of sputum mrna and CFU in patients receiving INH monotherapy showed icl mrna to be present in highest numbers at baseline and throughout the 7-day course of treatment (Figure 1). Among the molecular markers measured, icl mrna correlated the strongest with CFU at baseline, based on the intercept values (p<0.002) (Table 2). Both icl and hspx mrna had the highest correlation with the CFU decline from baseline to 7 days of therapy (p<0.02), as determined by repeated measure analysis of the change in the slopes (Table 2). RrnA-P1 mrna and fbpb mrna correlated significantly with CFU values at baseline and over the 7 days of therapy, but to a lesser extent than icl and hspx mrna. Classical EBA studies measure changes in quantitative colony counts between baseline and day 2 of therapy as a marker of bactericidal activity and the slope of decline between days 2 and 7 as a measure of sterilizing activity. We therefore performed analyses using these same time points and found no correlation between any of the measured mrna markers and the CFU between baseline and day 2. The 0-2 day EBA of 0.67 log10 CFU/ml/day for INH reported by Johnson et al. (9) was as expected from previous studies (3). None of the molecular markers declined appreciably during the same time with exception of fbpb mrna which decreased 0.47 log10 molecules/ml/day. Examining the slopes of decline from day 2 to day 7, there was a statistically significant correlation between both icl (r=.66, p<.04) and rrna-p1 (r=.78, p<.008) transcripts and CFU counts; this correlation did not hold for fbpb and hspx. Clearance of sputum icl mrna in response to fluoroquinolone monotherapy.

7 Serially collected sputum specimens from patients receiving monotherapy with one of three fluoroquinolones were tested for icl mrna levels (Table 3). The mean icl mrna levels were the same for each of the treatment groups and within one log10 of the mean for the INH group. As seen with the INH group, there was a significant correlation with CFU counts at baseline and decline from baseline to day 7 in each of the fluoroquinolone groups. There was no difference between the three fluoroquinolones regarding these correlations, which is consistent with the previously reported bactericidal activity between days 2 and 7 for all three fluoroquinolones (9). Therefore, results for the fluoroquinolones groups were combined into one group for comparison to the INH group. The decline in mrna levels from baseline to day 7 was similar between the two groups (Figure 2). These findings differ from previously published data showing that in a pooled comparison the EBA 2-7 of these fluoroquinolones was greater than that of INH (9). Clearance of sputum icl mrna in response to a rifampin-based standard drug regimen. To determine if icl mrna levels were detectable following one and two months of a rifampin-based drug regimen we randomly selected 20 patients receiving standard short-course chemotherapy (2 months of daily INH, rifampin, ethambutol and pyrazinamide followed by 4 months of daily INH and rifampin) as part of a larger adjunctive IL-2 study (10). Among 38 sputum specimens tested (19 patients with duplicate specimens) all with corresponding positive cultures on Middlebrook 7H10 agar and in BACTEC B medium at one month, 100% had detectable icl mrna. At two months, of 38 samples tested (two patients had only one specimen), 6 had growth on 7H10 agar (16%), 25 (66%) had growth in 12B medium, and 7 were positive for icl mrna (18%). At 2 months icl mrna results and liquid culture correlated more strongly (r=.34, p=0.04) than did growth on solid media with that of liquid culture (r=.16, p=0.34) (Table 4), suggesting that icl mrna is at least equally sensitive and specific as solid culture at two months, using growth in liquid culture as comparator. Normalizing mrna levels to examine gene expression during course of monotherapy.

8 An ideal molecular marker of response to therapy that reflects CFU in sputum should not change its expression level when M. tuberculosis is exposed to anti-tb drugs or the host immune response. To examine this, all longitudinal data for icl, fbpb, and hspx mrna from the INH and moxifloxacin monotherapy arms were normalized to rrna-p1 mrna, the product of which has been shown to contribute at a steady level to precursor mrna synthesis regardless of the stage of growth (11, 14). The ratios of icl, fbpb, and hspx mrna to rrna-p1 transcripts were consistent from baseline to 7 days of treatment and between the two drugs (mean values of the ratios, INH vs. moxifloxacin: icl, 1.27 vs. 1.36; hspx, 0.77 vs. 0.80; fbpb, 0.72 vs. 0.77) indicating that expression levels for these genes remained steady during the first 7 days of treatment, irrespective of the class of drug administered. Discussion Molecular markers of M. tuberculosis viability are attractive since results are rapid and there is potential for great analytical sensitivity. Previous studies have demonstrated that DNA assays are not useful in monitoring response to therapy since M. tuberculosis DNA persists well beyond the time points that cultures are positive, i.e., one to two months (2, 8, 7). This is likely due to continued shedding of intact dormant or dead tubercle bacilli from the focus of infection and due to an inherent resistance of DNA to degradation when sequestered within macrophages. Ribosomal RNA, which is present in higher amounts in the cell, is more labile than DNA; however, levels may decline slowly in the presence of effective drug therapy (13, 6). In our prior studies we demonstrated that fbpb mrna rapidly declines in response to standard short-course TB therapy and that its expression correlates with treatment outcome. By 14 days of therapy 13 of 19 patients no longer had detectable fbpb mrna in their sputum, even though cultures were still positive (2). The inability of the assay to detect fbpb mrna despite continued growth of M. tuberculosis in culture raised concern that the threshold of detection for this marker is too high. The ideal marker would be detectable as long as cultures remain positive and disappear when the tubercle bacilli are no longer

9 able to be cultured. Expanding on our previous studies we compared four different M. tuberculosis mrna targets in the context of an EBA study comparing INH with three newer fluoroquinolones (9). Icl mrna was determined to be the best marker based on high levels of expression in sputum and strong correlation with CFU counts, both at baseline and during the seven days of INH monotherapy. Icl mrna was also highly correlated with CFU in patients receiving fluoroquinolone monotherapy. In addition, icl mrna was measurable in all culture-positive patients following one month of treatment with a standard 4-drug rifampin-containing regimen. At two months icl mrna more closely correlated with growth in BACTEC 12B liquid culture than did growth on 7H10 medium. Expression of Icl, a gating enzyme of the glyoxylate cyle, is upregulated during M. tuberculosis infection in mice and human patients (16, 5). Likewise, mrna expression of Acr (alpha crystalline protein) is upregulated upon infection of macrophages and is thought to be important for survival during the persistent phase of infection (19, 1). Levels of icl mrna were higher than hspx mrna in patients prior to treatment and during monotherapy, therefore the threshold of detection was lower for icl mrna. This higher sensitivity enabled detection of icl mrna out to two months in liquid culture positive patients treated with a rifampin-containing regimen. This was in contrast to our previous observation that hspx mrna declined to undetectable levels within the first 14 to 28 days of therapy. In the EBA study, levels of icl, hspx, and fbpb mrna gradually decreased by approximately 1 log10 from baseline to day 7 in patients receiving INH monotherapy. In contrast the CFUs decreased 1.2 logs10 during the first two days and another 0.5 log10 over the next five days, demonstrating the typical biphasic killing curve of INH. A decline of 0.94 log10 was seen with fbpb mrna from baseline to day 2; however, there was not a significant correlation between change in fbpb mrna from baseline to day 2 and the 0-2 day EBA (CFU). The similar patterns of decline suggest that fbpb mrna may be useful in early bactericidal activity evaluations of individual anti-tb drugs and

10 further study of this marker in EBA studies is warranted. Although the data suggest that icl mrna may not be a useful marker of early bactericidal activity, the high correlation between icl mrna and CFU for the 2-7 day period provides additional evidence that it is a measure of sterilizing activity. RrnA-P1, one of five rrn promoters in mycobacteria, has been described as a novel RNA standard in analysis of quantitative RT-PCR transcriptional data (11, 14). In contrast to siga and rrs (16S rrna), rrna-p1 does not change during different growth phases and is present in similar amounts as any mrna in the cell. Normalizing values of icl, hspx, and fbpb mrna with rrna-p1 mrna demonstrated that expression of these genes was unaffected during the first seven days of single drug therapy. It is also likely that regulation of these genes was not influenced by the standard 4-drug therapy since mrna levels continued to decline steadily similar to that observed for CFU during the intensive phase of TB treatment. Limitations of our study were small sample size with limited power to detect small differences between groups. Strengths were frequent sputum cultures and intensive follow-up; supervised TB treatment and specimen collection; blinding of laboratory staff performing quantitative sputum cultures and mrna assays to treatment assignment; and the comparison of patients treated with drugs with different mechanisms of action: INH inhibition of mycolic acid and cell wall synthesis; rifampin inhibition of transcription by binding to DNA-dependent RNA polymerase; and fluoroquinolones inhibition of DNA gyrase. This study provides additional support for analyzing sputum mrna by quantitative RT-PCR to monitor response to therapy and evaluate the bacteriologic activity of promising individual drugs during the early clinical testing. In particular icl mrna shows promise as a replacement for quantitative culture (CFU counts) in the evaluation of new TB regimens. In addition, icl mrna could potentially serve as a surrogate marker for long-term treatment response. Both of these applications will require larger longitudinal studies to validate the reliability of icl mrna as

11 248 surrogate marker of response to TB therapy Acknowledgements This work was supported by the Tuberculosis Research Unit at Case Western Reserve University, established with funds from the United States National Institutes of Allergy and Infectious Diseases, National Institutes of Health and Human Services, under Contract No. NO1-AI95383 and HHSN C/NO1-AI Clinical trial registration number: NCT The authors thank the patients and staff of the Tuberculosis Clinic and Clinical Research Center of the Hospital Universitário Cassiano Antônio de Moraes and the Núcleo de Doenças Infecciosas (NDI) of UFES, Vitória, Brazil; the Ugandan National Tuberculosis Treatment Center, Mulago Hospital and the Ugandan National Tuberculosis and Leprosy Programme; and the TB laboratories of the Joint Clinical Research Centre, Kampala, Uganda and UFES NDI for their invaluable help with the original clinical trials on which the current study is based. References 1. Desjardin, L. E., Hayes, L. G., Sohaskey, C. D., Wayne, L. G., and K. D. Eisenach Microaerophilic induction of the alpha-crystallin chaperone protein homologue (hspx) mrna of Mycobacterium tuberculosis. J. Bacteriol.183: Desjardin, L. E., Perkins, M. D., Wolski. K., Haun, S., Teixeira, L., Chen, Y., Johnson, J.L., Ellner, J., Dietze, R., Bates, J., Cave, M. D., and K. D. Eisenach Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy. Am. J. Respir. Crit. Care Med. 160: Dietze, R., Teixeira, L., Rocha, L. M., Palaci, M., Johnson, J. L., Wells, C., Rose, L., Eisenach, K., and J. J. Ellner Safety and bactericidal activity of rifalazil in patients with pulmonary tuberculosis. Antimicrob. Agents Chemother. 45:

12 Donald, P. R., Sirgel, F. A., Venter, A., Parkin, D. P., Seifart, H. I., van de Wal, B. W., Maritz, J. S., and P. B. Fourie Early bactericidal activity of antituberculosis agents. Expert Rev. Anti Infect. Ther. 1: Erol, A Visceral adipose tissue specific persistence of Mycobacterium tuberculosis may be reason for the metabolic syndrome. Med. Hypotheses. 71: Gamboa, F., Manterola, J. M., Lonca, J., Vinado, B., Matas, L., Gimenez, M., Manzano, J. R., Rodrigo, C., Cardona, P. J., Padilla, E., Dominguez, L., and V. Ausina Rapid detection of Mycobacterium tuberculosis in respiratory specimens, blood and other non-respiratory specimens by amplification of rrna. Int J Tuberc Lung Dis 1: Hellyer, T. J., DesJardin, L. E., Assaf, M. K., Bates, J. H., Cave, M. D., and K. D. Eisenach Specificity of IS6110-based amplification assays for Mycobacterium tuberculosis complex. J. Clin. Microbiol. 34: Hellyer, T. J., DesJardin, L. E., Hehman, G. L., Cave, M. D., and K. D. Eisenach Quantitative analysis of mrna as a marker for viability of Mycobacterium tuberculosis.. J. Clin. Microbiol. 37: Johnson, J. L., Hadad, D. J., Boom, W. H., Daley, C. L., Peloquin, C. A., Eisenach, K. D., Jankus, D. D., Debanne, S. M., Charlebois, E. D., Maciel, E., Palaci, M., and R. Dietze Early and extended early bactericidal activity of levofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis. Int. J. Tuberc. Lung Dis. 10: Johnson, J. L., Ssekasanvu, E., Okwera, A., Mayanja, H., Hirsch, C. S., Nakibali, J. G., Jankus, D. D., Eisenach, K. D., Boom,W. H., Ellner, J. J., and R. D. Mugerwa Randomized trial of adjunctive interleukin-2 in adults with pulmonary tuberculosis. Am. J. Respir. Crit. Care Med. 168: Menendez, M. C., Garcia, M. J., Navarro, M. C., Gonzalez-y-Merchand, J. A., Rivera- Gutierrez, S., Garcia-Sanchez, L., and R. A. Cox Characterization of an rrna operon

13 (rrnb) of Mycobacterium fortuitum and other mycobacterial species: implications for the classification of mycobacteria. J. Bacteriol. 184: Mitchison, D.A Assessment of new sterilizing drugs for treating pulmonary tuberculosis by culture at 2 months. Am. Rev. Respir. Dis. 147: Moore, D.F., Curry, J.I., Knott, C.A., and V. Jonas Amplification of rrna for assessment of treatment response of pulmonary tuberculosis patients during antimicrobial therapy. J. Clin. Microbiol. 34: Nunez, M. C., Menendez, M. C., Rebollo, M. J., and M. J. Garciav Transcriptional analysis of Mycobacterium fortuitum cultures upon hydrogen peroxide treatment using the novel standard rrna-p1. BMC Microbiol. 8: Ribeiro-Rodrigues, R., Resende Co, T., Johnson, J. L., Ribeiro, F., Palaci, M., Sa, R. T., Maciel, E. L., Pereira Lima, F. E., Dettoni, V., Toossi, Z., Boom, W. H., Dietze, R., Ellner, J. J., and C. S. Hirsch Sputum cytokine levels in patients with pulmonary tuberculosis as early markers of mycobacterial clearance. Clin. Diagn. Lab. Immunol. 9: Timm, J., Post, F. A., Bekker, L. G., Walther, G. B., Wainwright, H. C., Manganelli, R., Chan, W. T., Tsenova, L., Gold, B., Smith, I., Kaplan, G., and J. D. McKinney Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients. Proc. Natl. Acad. Sci. USA. 100: Wallis. R. S., and J. L. Johnson The role of surrogate markers in the clinical evaluation of anti-tuberculous chemotherapy. Current Medicinal Chemistry: Anti-Infective Agents. 4: Wallis, R. S., Vinhas, S., Johnson, J. L., Ribeiro, F. C., Palaci, M., Peres, R. L., Sa, R. T., Dietze, R., Chiunda, A., Eisenach, K., and J. J. Ellner Whole blood bactericidal activity during treatment of pulmonary tuberculosis. J. Infect. Dis. 187: Yuan, Y., Crane, D. D., Simpson, R. M., Zhu, Y. Q., Hickey, M. J., Sherman, D. R., Barry 3 rd, C. E The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is

14 323 required for growth in macrophages. Proc. Natl. Acad. Sci. USA. 95:

15 325 Figure Legends Figure 1. Decline in Mtb mrna and CFU in sputum from patients with TB during 7 days of INH monotherapy. Sputum was collected during a 12 h time period for 2 days before and then daily during 7 days of INH administration. (Time 0 is the mean of the two samples prior to INH). Data are mean values in molecules of mrna or CFU/ml (log10) of sputum + SD for each time interval. Icl, isocitrate lyase; rrna-p1, non-coding ribosomal promoter region; hspx, alpha crystalline protein; fbpb, fibronectin-binding protein antigen 85B; CFU, colony forming units; SD, standard deviation. Figure 2. Decline in icl mrna levels in sputum from patients with TB receiving INH monotherapy for 7 days compared with the decline in icl mrna in sputum from patients receiving fluoroquinolone monotherapy (results combined for the three groups on moxifloxacin, gatifloxacin, and levofloxacin). Data are mean values in molecules of mrna/ml (log10) of sputum + SD for each time interval. FQ, fluoroquinolones; icl, isocitrate lyase; SD, standard deviation.

16 339 Table 1. Sequences of primers and probes used for quantification of specific RNA products Target RT and PCR primer sequences (5 to 3 )a Taqman probe sequence (5 to 3 )b fbpb F: CTC GGA ATT CGC GTA CGG R: TCG TCA GCA CCT ACC GGC CCT TCG TTC GCA CGG TGT CGC RT primer: TCC TGG AAC TTC AGG TTG CT hspx F: TTA TTG CGG GAA CGG CA CAC CGC CCA ACT CGT TCG GG R: AGA ACT CGG CGG GTA TGT TG RT primer: CGA CAA GGA CGT CGA CAT TA icl1 F: CAC ATC CGC ACT TTG ACG TC CTC GGC TCG CGG CCG ATG T R: ATC ACC ACC GTG GGA ACA TC RT primer: GTT CTT GGT GCG GTA GAA GC rrna-p1 F: CCT ATG GAT ATC TAT GGA TGA CCG A CCT GGT CTT GAC TCC ATT GCC GGA R: GCA ACC CTG CCA GTC TAA TAC AA RT primer: TTC TCA AAC AAC ACG CTT GC a F, forward; R, reverse b Probes labeled on 5 end with 5-carboxyfluoroscein (FAM) and N,N,N,N -tetramethyl-6- carboxyrhodamine (TAMARA) on the 3 end.

17 Table 2. Correlations between the mean intercepts (baseline values) and mean slopes (baseline to 7 day values) of log10 molecules of mrna/ml and log10 CFU/ml of sputum for the four M. tuberculosis molecular markers in 10 patients receiving INH monotherapy mrna Target Mean mrna at baseline a Correlation of mrna & CFU at baseline (SEM b ) Intercept P value Mean mrna for slope from baseline to day 7 Correlation of mrna & CFU slopes baseline to day 7 (SEM) Slope P value icl (0.196) (0.041) 0.02 rrna-p (0.241) (0.040) 0.04 hspx (0.176) (0.037) 0.02 fbpb (0.220) (0.035) 0.04 a Mean baseline values were based on 10 patients each having 2 specimens collected in the 2 days before initiation of therapy. b SEM, standard error of mean.

18 Table 3. Correlation of icl mrna with CFU at baseline and over the 7-day course of therapy by assigned fluoroquinolone monotherapy versus INH 353 Mean icl mrna/ml at baseline Mean slope between baseline and (correlation P value day 7 P value Number patients with CFU/ml) SEM for baseline (correlation with CFU) SEM for slope Isoniazid (0.86) (0.71) Levofloxacin (0.90) (0.86) Gatifloxacin (0.68) (0.95) <.0001 Moxifloxacin (0.71) (0.75)

19 Table 4. Liquid, solid, and icl mrna results at 1 and 2 months while on standard 4 drug therapy and the correlation between each of these methods. Liquid culture (BACTEC B) Solid culture (7H10) icl mrna Month 1 (n = 38) a Month 2 (n=38) Correlations (Month 2) R value P value Liquid and solid Liquid and icl mrna b Solid and icl mrna a all methods with correlation of R=1 at 1 month, unable to calculate p value. b value of statistical significance

20 molecules of mrna/ml sputum or CFU/ml sputum (log10) Figure icl 9 8 rrna-p1 7 CFU 6 5 hspx 4 fbpb Days of INH monotherapy

21 molecules of ICL mrna/ml sputum (log10) Figure FQ INH Days on monotherapy