Changes in Tuberculosis Blood RNA Signatures During the Short-course Treatment of Rifampicin-resistant Tuberculosis

Abstract Introduction: Blood RNA signatures have been widely explored for distinguishing healthy individuals and those with latent tuberculosis infection from patients with active tuberculosis, but their changes during the treatment process have been rarely studied, especially in the context of drug-resistant tuberculosis treatment. Methods: This study was a sub-study embedded within the TB-TRUST study (NCT03867136) and TB-TRUST plus study (NCT04717908). Participants aged 18-70 years old with pulmonary rifampicin-resistant tuberculosis were prospectively enrolled and treated with the all-oral shorter regimens or the standard injectable-containing shorter regimen. We collected peripheral blood samples from participants before treatment (pre-treatment), at the first negative smear (smear conversion), and at the end of treatment (treatment completion). Transcriptome sequencing was performed using the Illumina NovaSeq platform with PE150. Quality control and quantification of the sequencing results were conducted by fastp, STAR, FeatureCount and StringTie, converting the results into TPM format. Twelve previously reported blood RNA signatures were included in the assessment and standardized by Z-scores. The Wilcoxon rank-sum test was conducted to compare the changes in cores across pre-treatment, smear conversion, and treatment completion for the 12 signatures. Results: A total of 32 patients with positive smear were recruited in the study, including 17 patients receiving standard injectable-containing shorter regimen and 15 patients receiving all-oral shorter regimens. Patients provided 76 peripheral blood samples at the required time points (25 at pre-treatment, 26 at smear conversion, and 25 at treatment completion). Twenty-seven patients in this study achieved favorable outcome, and five discontinued treatment for non-bacteriological reasons. The median time to smear conversion was 3.5 weeks and median duration of treatment was 36.5 weeks. Samples from 19 patients were collected at all three time points. No significant differences were observed in the 12 blood RNA signatures at smear conversion compared to pre-treatment. Most blood RNA biomarkers showed a significant decrease at post-treatment compared to pre-treatment, with Gliddon3 (p=2.5×10-5), BATF2 (p=3.7×10-5), and Darboe11 (p=2.6×10-4) being the three most significantly changed scores. In subgroup analysis, no differences in 12 scores were observed between two regimens at three time points. Conclusions: In drug-resistant tuberculosis patients who completed treatment, tuberculosis blood RNA signatures showed significant differences compared to baseline. However, included tuberculosis blood RNA signatures did not adequately reflect contemporary host microbial clearance in the early stage of treatment.