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mRNA 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 tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (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. mRNA 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 CFU 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.
The development of new drugs for tuberculosis (TB) treatment has been hampered by the lack of an early surrogate marker that reflects nonrelapsing 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 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 (2, 17), 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 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-PCR (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 versus 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 short-course chemotherapy in a trial of adjunctive interleukin-2 (IL-2) in TB treatment.
As previously described, 40 Brazilian adults with smear-positive fully drug susceptible TB were randomly assigned to receive 7 days of daily isoniazid (INH) at 300 mg, levofloxacin at 1,000 mg, gatifloxacin at 400 mg, or moxifloxacin at 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 numbers of CFU/ml per sample as previously described (3). One-half-milliliter aliquots of N-acetyl-l-cysteine (NALC)-digested sputum were frozen at −70°C until RNA extraction.
One hundred 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 medium and quantitative culture on Middlebrook 7H10 agar plates. One-half-milliliter aliquots of NALC-digested sputum were stored at −70°C until RNA extraction. For the current substudy, 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.
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 (Table1),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 102 to 105 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.
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× TaqMan universal PCR master mix (ABI), 0.9 μM each primer, and 0.25 μM probe (Table (Table1).1). The quantity of specific target DNA was determined from the threshold cycle (CT) value with reference to a standard curve of genomic DNA (5 to 78,125 copies) obtained from Mycobacterium bovis ATCC 19210. The graphs of starting DNA concentration versus CT value were consistently linear (R2 = 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.
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.
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 6.52 ± 0.66 CFU/ml of sputum at baseline for all groups 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 (Table22).
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 (Fig. (Fig.1).1). Among the molecular markers measured, icl mRNA correlated the strongest with CFU at baseline, based on the intercept values (P < 0.002) (Table (Table2).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 (Table2).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- to 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 the exception of fbpB mRNA, which decreased 0.47 log10 molecules/ml/day. In examining the slopes of decline from day 2 to day 7, there was a statistically significant correlation between both icl (r = 0.66, P < 0.04) and rrnA-P1 (r = 0.78, P < 0.008) transcripts and CFU counts; this correlation did not hold for fbpB and hspX.
Serially collected sputum specimens from patients receiving monotherapy with one of three fluoroquinolones were tested for icl mRNA levels (Table (Table3).3). The mean icl mRNA levels were the same for each of the treatment groups and within 1 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 fluoroquinolone 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 (Fig. (Fig.2).2). These findings differ from previously published data showing that in a pooled comparison the EBA at days 2 to 7 of these fluoroquinolones was greater than that of INH (9).
To determine if icl mRNA levels were detectable following 1 and 2 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 460 12B medium at 1 month, 100% had detectable icl mRNA (Table (Table4).4). At 2 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 = 0.34, P = 0.04) than did growth on solid medium with that of liquid culture (r = 0.16, P = 0.34) (Table (Table5),5), suggesting that icl mRNA is at least equally as sensitive and specific as solid culture at 2 months, using growth in liquid culture as comparator.
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 versus moxifloxacin: icl, 1.27 versus 1.36; hspX, 0.77 versus 0.80; fbpB, 0.72 versus 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.
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., 1 to 2 months (2, 7, 8). 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. rRNA, which is present in larger amounts in the cell, is more labile than DNA; however, levels may decline slowly in the presence of effective drug therapy (6, 13). 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 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 7 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 1 month of treatment with a standard 4-drug rifampin-containing regimen. At 2 months icl mRNA more closely correlated with growth in Bactec 12B liquid culture than did growth on 7H10 medium.
Expression of the icl product, a gating enzyme of the glyoxylate cycle, is upregulated during M. tuberculosis infection in mice and human patients (5, 16). 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 (1, 19). 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 2 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 CFU decreased 1.2 log10 during the first 2 days and another 0.5 log10 over the next 5 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- to 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 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- to 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 amounts similar to those of 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 7 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 a surrogate marker of response to TB therapy.
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 HHSN266200700022C/NO1-AI-70022.
The clinical trial registration number is NCT00396084.
We 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.
Published ahead of print on 18 November 2009.