Transcriptional adaptation of Mycobacterium tuberculosis that survives prolonged multi-drug treatment in mice.

To address the ongoing global tuberculosis crisis, there is a need for shorter, more effective treatments. A major reason why tuberculosis requires prolonged treatment is that, following a short initial phase of rapid killing, the residual Mycobacterium tuberculosis withstands drug killing. Because existing methods lack sensitivity to quantify low-abundance mycobacterial RNA in drug-treated animals, cellular adaptations of drug-exposed bacterial phenotypes in vivo remain poorly understood. Here, we used a novel RNA-seq method called SEARCH-TB to elucidate the Mycobacterium tuberculosis transcriptome in mice treated for up to 28 days with standard doses of isoniazid, rifampin, pyrazinamide, and ethambutol. We compared murine results with in vitro SEARCH-TB results during exposure to the same regimen. Treatment suppressed genes associated with growth, transcription, translation, synthesis of rRNA proteins, and immunogenic secretory peptides. Bacteria that survived prolonged treatment appeared to transition from ATP-maximizing respiration toward lower-efficiency pathways and showed modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pump expression. Although the pre-treatment in vivo and in vitro transcriptomes differed profoundly, genes differentially expressed following treatment in vivo and in vitro were similar, with differences likely attributable to immunity and drug pharmacokinetics in mice. These results reveal cellular adaptations of Mycobacterium tuberculosis that withstand prolonged drug exposure in vivo, demonstrating proof of concept that SEARCH-TB is a highly granular pharmacodynamic readout. The surprising finding that differential expression is concordant in vivo and in vitro suggests that insights from transcriptional analyses in vitro may translate to the mouse. IMPORTANCE A major reason that curing tuberculosis requires prolonged treatment is that drug exposure changes bacterial phenotypes. The physiologic adaptations of Mycobacterium tuberculosis that survive drug exposure in vivo have been obscure due to low sensitivity of existing methods in drug-treated animals. Using the novel SEARCH-TB RNA-seq platform, we elucidated Mycobacterium tuberculosis phenotypes in mice treated for with the global standard 4-drug regimen and compared them with the effect of the same regimen in vitro. This first view of the transcriptome of the minority Mycobacterium tuberculosis population that withstands treatment in vivo reveals adaptation of a broad range of cellular processes, including a shift in metabolism and cell wall modification. Surprisingly, the change in gene expression induced by treatment in vivo and in vitro was largely similar. This apparent "portability" from in vitro to the mouse provides important new context for in vitro transcriptional analyses that may support early preclinical drug evaluation.

Transcriptional adaptation of drug-tolerant Mycobacterium tuberculosis in mice.

Transcriptome evaluation of Mycobacterium tuberculosis in the lungs of laboratory animals during long-term treatment has been limited by extremely low abundance of bacterial mRNA relative to eukaryotic RNA. Here we report a targeted amplification RNA sequencing method called SEARCH-TB. After confirming that SEARCH-TB recapitulates conventional RNA-seq in vitro, we applied SEARCH-TB to Mycobacterium tuberculosis-infected BALB/c mice treated for up to 28 days with the global standard isoniazid, rifampin, pyrazinamide, and ethambutol regimen. We compared results in mice with 8-day exposure to the same regimen in vitro. After treatment of mice for 28 days, SEARCH-TB suggested broad suppression of genes associated with bacterial growth, transcription, translation, synthesis of rRNA proteins and immunogenic secretory peptides. Adaptation of drug-stressed Mycobacterium tuberculosis appeared to include a metabolic transition from ATP-maximizing respiration towards lower-efficiency pathways, modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pumps expression. Despite markedly different expression at pre-treatment baseline, murine and in vitro samples had broadly similar transcriptional change during treatment. The differences observed likely indicate the importance of immunity and pharmacokinetics in the mouse. By elucidating the long-term effect of tuberculosis treatment on bacterial cellular processes in vivo, SEARCH-TB represents a highly granular pharmacodynamic monitoring tool with potential to enhance evaluation of new regimens and thereby accelerate progress towards a new generation of more effective tuberculosis treatment.

A comparison of methods for multiple degree of freedom testing in repeated measures RNA-sequencing experiments.

BACKGROUND: As the cost of RNA-sequencing decreases, complex study designs, including paired, longitudinal, and other correlated designs, become increasingly feasible. These studies often include multiple hypotheses and thus multiple degree of freedom tests, or tests that evaluate multiple hypotheses jointly, are often useful for filtering the gene list to a set of interesting features for further exploration while controlling the false discovery rate. Though there are several methods which have been proposed for analyzing correlated RNA-sequencing data, there has been little research evaluating and comparing the performance of multiple degree of freedom tests across methods. METHODS: We evaluated 11 different methods for modelling correlated RNA-sequencing data by performing a simulation study to compare the false discovery rate, power, and model convergence rate across several hypothesis tests and sample size scenarios. We also applied each method to a real longitudinal RNA-sequencing dataset. RESULTS: Linear mixed modelling using transformed data had the best false discovery rate control while maintaining relatively high power. However, this method had high model non-convergence, particularly at small sample sizes. No method had high power at the lowest sample size. We found a mix of conservative and anti-conservative behavior across the other methods, which was influenced by the sample size and the hypothesis being evaluated. The patterns observed in the simulation study were largely replicated in the analysis of a longitudinal study including data from intensive care unit patients experiencing cardiogenic or septic shock. CONCLUSIONS: Multiple degree of freedom testing is a valuable tool in longitudinal and other correlated RNA-sequencing experiments. Of the methods that we investigated, linear mixed modelling had the best overall combination of power and false discovery rate control. Other methods may also be appropriate in some scenarios.

Comparison of single time-point [111-In] pentetreotide SPECT/CT with dual time-point imaging of neuroendocrine tumors.

PURPOSE: to determine whether single time-point single-photon emission computed tomography-computed tomography (SPECT/CT) somatostatin receptor imaging can replace traditional dual time-point planar and SPECT somatostatin receptor scintigraphy for evaluation of neuroendocrine tumors. MATERIALS AND METHODS: twenty-four patients (9 males, 15 females; mean age: 56 years; range: 14-82 years) underwent [111-In] pentetreotide scintigraphy, with planar whole-body images acquired at 24 and 48 hours after injection and abdominal SPECT/CT at 24 hours postinjection. Two blinded readers independently interpreted each study, using single time-point (24 hours planar and SPECT/CT) and separately using dual time-point (24- and 48-hours planar, and 24-hour SPECT without CT) image information. Consensus interpretations were compared with surgical pathology, or clinical and radiologic follow-up for at least 12 months. RESULTS: Interobserver agreement was excellent (κ = 0.86) for single time-point imaging, and good (κ = 0.56) with dual time-point imaging. After consensus review, single time-point imaging identified pathologic lesions in 11 of 12 subjects with diagnosis of NET at follow-up, and in 0 of 12 subjects without NET (sensitivity 92%; specificity 100%). Dual time-point imaging performed similarly, but missed an additional NET case (sensitivity 83%; specificity 100%). After review of SPECT/CT, the readers considered that additional 48 hours imaging was not necessary in the majority of cases, indicating high degree of confidence with the single time-point imaging. CONCLUSION: [111-In] pentetreotide SPECT/CT imaging at 24 hours identifies pathologic disease sites and distinguishes physiologic activity equally well compared to traditional strategies using 2 imaging days. Routine use of SPECT/CT will allow single time-point imaging without loss of diagnostic accuracy, enhancing patient convenience, and clinical throughput.