In silico evaluation and exploration of antibiotic tuberculosis treatment regimens

Abstract Background Improvement in tuberculosis treatment regimens requires selection of antibiotics and dosing schedules from a large design space of possibilities. Incomplete knowledge of antibiotic and host immune dynamics in tuberculosis granulomas impacts clinical trial design and success, and variations among clinical trials hamper side-by-side comparison of regimens. Our objective is to systematically evaluate the efficacy of isoniazid and rifampin regimens, and identify modifications to these antibiotics that improve treatment outcomes. Results We pair a spatio-temporal computational model of host immunity with pharmacokinetic and pharmacodynamic data on isoniazid and rifampin. The model is calibrated to plasma pharmacokinetic and granuloma bacterial load data from non-human primate models of tuberculosis and to tissue and granuloma measurements of isoniazid and rifampin in rabbit granulomas. We predict the efficacy of regimens containing different doses and frequencies of isoniazid and rifampin. We predict impacts of pharmacokinetic/pharmacodynamic modifications on antibiotic efficacy. We demonstrate that suboptimal antibiotic concentrations within granulomas lead to poor performance of intermittent regimens compared to daily regimens. Improvements from dose and frequency changes are limited by inherent antibiotic properties, and we propose that changes in intracellular accumulation ratios and antimicrobial activity would lead to the most significant improvements in treatment outcomes. Results suggest that an increased risk of drug resistance in fully intermittent as compared to daily regimens arises from higher bacterial population levels early during treatment. Conclusions Our systems pharmacology approach complements efforts to accelerate tuberculosis therapeutic development.http://deepblue.lib.umich.edu/bitstream/2027.42/116019/1/12918_2015_Article_221.pd

Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers

Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers

A Model of Tuberculosis Transmission and Intervention Strategies in an Urban Residential Area

The model herein aims to explore the dynamics of the spread of tuberculosis (TB) in an informal settlement or township. The population is divided into households of various sizes and also based on commuting status. The model dynamics distinguishes between three distinct social patterns: the exposure of commuters during travel, random diurnal interaction and familial exposure at night. Following the general SLIR models, the population is further segmented into susceptible (S), exposed/latently infected (L), active/infectious (I), and recovered (R) individuals. During the daytime, commuters travel on public transport, while non-commuters randomly interact in the community to mimic chance encounters with infectious persons. At night, each family interacts and sleeps together in the home. The risk of exposure to TB is based on the proximity, duration, and frequency of encounters with infectious persons. The model is applied to a hypothetical population to explore the effects of different intervention strategies including vaccination, wearing of masks or scarves during the commute, prophylactic treatment of latent infections and more effective case-finding and treatment. The most important findings of the model are: (1) members of larger families are responsible for more disease transmissions than those from smaller families, (2) daily commutes on public transport provide ideal conditions for transmission of the disease, (3) improved diagnosis and treatment has the greatest impact on the spread of the disease, and (4) detecting TB at the first clinic visit, when patients are still smear negative, is key

A Model of Tuberculosis Transmission and Intervention Strategies in an Urban Residential Area

The model herein aims to explore the dynamics of the spread of tuberculosis (TB) in an informal settlement or township. The population is divided into households of various sizes and also based on commuting status. The model dynamics distinguishes between three distinct social patterns: the exposure of commuters during travel, random diurnal interaction and familial exposure at night. Following the general SLIR models, the population is further segmented into susceptible (S), exposed/latently infected (L), active/infectious (I), and recovered (R) individuals. During the daytime, commuters travel on public transport, while non-commuters randomly interact in the community to mimic chance encounters with infectious persons. At night, each family interacts and sleeps together in the home. The risk of exposure to TB is based on the proximity, duration, and frequency of encounters with infectious persons. The model is applied to a hypothetical population to explore the effects of different intervention strategies including vaccination, wearing of masks or scarves during the commute, prophylactic treatment of latent infections and more effective case-finding and treatment. The most important findings of the model are: (1) members of larger families are responsible for more disease transmissions than those from smaller families, (2) daily commutes on public transport provide ideal conditions for transmission of the disease, (3) improved diagnosis and treatment has the greatest impact on the spread of the disease, and (4) detecting TB at the first clinic visit, when patients are still smear negative, is key

Experimental Validation of a Fundamental Model for PCR Efficiency

Recently a theoretical analysis of PCR efficiency has been published by Booth et al., (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii)the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii)the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. Experiments have been designed that are specifically controlled by each one of the efficiencies and the results are consistent with the mathematical model. The experimental data has also been used to quantify six key parameters of the theoretical model. An important application of the fully characterized model is to calculate initial template concentration from real-time PCR data. Given the PCR protocol, the midpoint cycle number (where the template concentration is half that of the final concentration) can be theoretically determined and graphed for a variety of initial DNA concentrations. Real-time results can be used to calculate the midpoint cycle number and consequently the initial DNA concentration, using this graph. The application becomes particularly simple if a conservative PCR protocol is followed where only the annealing efficiency is controlling

Efficiency of the Polymerase Chain Reaction

The polymerase chain reaction (PCR) has found wide application in biochemistry and molecular biology such as gene expression studies, mutation detection, forensic analysis and pathogen detection. Increasingly quantitative real time PCR is used to assess copy numbers from overall yield. In this study the yield is analyzed as a function of several processes: (1) thermal damage of the template and polymerase occurs during the denaturing step, (2) competition exists between primers and templates to either anneal or form dsDNA, (3) polymerase binding to annealed products (primer/ssDNA) to form ternary complexes and (4) extension of ternary complexes. Explicit expressions are provided for the efficiency of each process, therefore reaction conditions can be directly linked to the overall yield. Examples are provided where different processes play the yield-limiting role. The analysis will give researchers a unique understanding of the factors that control the reaction and will aid in the interpretation of experimental results

Fluoroquinolone Efficacy against Tuberculosis Is Driven by Penetration into Lesions and Activity against Resident Bacterial Populations.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of "universal" drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens

Rapid Diagnosis of Tuberculosis in a Peripheral Setting

Tuberculosis is an ancient and worldwide epidemic affecting millions of people in mainly the developing world, killing almost 2 million people in 2008. Current diagnostic techniques are outdated and have proven insufficient to control the disease. Smear microscopy has poor sensitivity and culture is slow to yield results. Modern diagnostic techniques are making great strides in shortening time to result but are restricted by two qualities: 1) prohibitively high costs prevent implementation in resource poor areas, and 2) equipment and technician requirements limit application to centralized laboratories. There exists a divide between new technologies and the people that need them most. Here, a novel epidemiological model of tuberculosis in an urban community confirms the importance of improved diagnostics in lowering prevalence. The model highlights the importance of sensitivity and accessibility. This work presents the development of a nucleic acid amplification test for tuberculosis diagnosis from sputum. The prototype system consists of 1) a sputum processing unit capable of extracting DNA within 5 minutes, and 2) a rapid PCR thermocycler which amplifies Mycobacterium tuberculosis complex specific sequences (IS6110 and IS1081) in under 15 minutes and detects product in real-time. Lysis protocol development was guided by a combined theoretical/experimental analysis of the kinetics of heat lysis of Mycobacterium smegmatis. The analysis revealed the activation energy of lysis (22.1 kcal/mole) and the minimum cell wall damage that result in cell distruction (14-17%). The PCR is capable of amplifying template amounts below smear microscopy concentrations. The test was applied to 58 clinical samples from the Steve Biko Academic Hospital in Pretoria, South Africa. Sensitivity was 95% on smear positive culture positive samples and 70% on smear negative culture positive samples. Specificity was 86%. In summary, the test moves toward an important niche of rapid (less than 30 minutes) and affordable ($5-10) diagnosis in a peripheral setting. Sensitivity of the test is comparable to other available systems, while specificity still needs improvement. However, turnaround times and costs are far below other tests currently being developed

The tri-frame model

The tri-frame model gives mathematical expression to the transcription and translation processes, and considers all three reading frames. RNA polymerases transcribe DNA in single nucleotide increments, but ribosomes translate mRNA in pairings of three (triplets or codons). The set of triplets in the mRNA, starting with the initiation codon (usually AUG) defines the open reading frame (ORF). Since ribosomes do not always translocate three nucleotide positions, two additional reading frames are accessible. The -1RF and the +1RF are triplet pairings of the mRNA which are accessed by shifting one nucleotide position in the 5’ and 3’ directions respectively. Transcription is modeled as a linear operator that maps the initial codons in all three frames into other codon sets to account for possible transcriptional errors. Translational errors (missense errors) originate from misacylation of tRNA’s and misreading of aa-tRNA’s by the ribosome. Translation is modeled as a linear mapping from codons into aa-tRNA species, which includes misreading errors. A final transformation from aa-tRNA species into amino acids provides the probability distributions of possible amino acids into which the codons in all three frames could be translated. An important element of the tri-frame model is the ribosomal occupancy probability. It is a vector in R3 that gives the probability to find the ribosome in the ORF, -1RF or +1RF at each codon position. The sequence of vectors, from the first to the final codon position, gives a history of ribosome frameshifting. The model is powerful: it provides exact expressions for: (1) yield of error-free protein, (2) fraction of prematurely terminated polypeptides, (3) number of transcription errors, (4) number of translation errors and (5) mutations due to frameshifting. The theory is demonstrated for the three genes rpsU, dnaG and rpoD of E. coli which lie on the same operon, as well as for the prfB gene