Optimizing mycobacteria molecular diagnostics: No decontamination! Human DNA depletion? Greener storage at 4 °C!

INTRODUCTION Tuberculosis (TB) is an infectious disease caused by the group of bacterial pathogens Mycobacterium tuberculosis complex (MTBC) and is one of the leading causes of death worldwide. Timely diagnosis and treatment of drug-resistant TB is a key pillar of WHO's strategy to combat global TB. The time required to carry out drug susceptibility testing (DST) for MTBC via the classic culture method is in the range of weeks and such delays have a detrimental effect on treatment outcomes. Given that molecular testing is in the range of hours to 1 or 2 days its value in treating drug resistant TB cannot be overstated. When developing such tests, one wants to optimize each step so that tests are successful even when confronted with samples that have a low MTBC load or contain large amounts of host DNA. This could improve the performance of the popular rapid molecular tests, especially for samples with mycobacterial loads close to the limits of detection. Where optimizations could have a more significant impact is for tests based on targeted next generation sequencing (tNGS) which typically require higher quantities of DNA. This would be significant as tNGS can provide more comprehensive drug resistance profiles than the relatively limited resistance information provided by rapid tests. In this work we endeavor to optimize pre-treatment and extraction steps for molecular testing. METHODS We begin by choosing the best DNA extraction device by comparing the amount of DNA extracted by five commonly used devices from identical samples. Following this, the effect that decontamination and human DNA depletion have on extraction efficiency is explored. RESULTS The best results were achieved (i.e., the lowest Ct values) when neither decontamination nor human DNA depletion were used. As expected, in all tested scenarios the addition of decontamination to our workflow substantially reduced the yield of DNA extracted. This illustrates that the standard TB laboratory practice of applying decontamination, although being vital for culture-based testing, can negatively impact the performance of molecular testing. As a complement to the above experiments, we also considered the best Mycobacterium tuberculosis DNA storage method to optimize molecular testing carried out in the near- to medium-term. Comparing Ct values following three-month storage at 4 °C and at -20 °C and showed little difference between the two. DISCUSSION In summary, for molecular diagnostics aimed at mycobacteria this work highlights the importance of choosing the right DNA extraction device, indicates that decontamination causes significant loss of mycobacterial DNA, and shows that samples preserved for further molecular testing can be stored at 4 °C, just as well at -20 °C. Under our experimental settings, human DNA depletion gave no significant improvement in Ct values for the detection of MTBC

Complete positivity and entangled degrees of freedom

We study how some recently proposed noncontextuality tests based on quantum interferometry are affected if the test particles propagate as open systems in presence of a gaussian stochastic background. We show that physical consistency requires the resulting markovian dissipative time-evolution to be completely positive.Comment: 23 pages, plain-TeX, no figure

Automatic Quantum Error Correction

Criteria are given by which dissipative evolution can transfer populations and coherences between quantum subspaces, without a loss of coherence. This results in a form of quantum error correction that is implemented by the joint evolution of a system and a cold bath. It requires no external intervention and, in principal, no ancilla. An example of a system that protects a qubit against spin-flip errors is proposed. It consists of three spin 1/2 magnetic particles and three modes of a resonator. The qubit is the triple quantum coherence of the spins, and the photons act as ancilla.Comment: 16 pages 12 fig LaTex uses multicol, graphicx expanded version of letter submitted to Phys Rev Let

'Return to equilibrium' for weakly coupled quantum systems: a simple polymer expansion

Recently, several authors studied small quantum systems weakly coupled to free boson or fermion fields at positive temperature. All the approaches we are aware of employ complex deformations of Liouvillians or Mourre theory (the infinitesimal version of the former). We present an approach based on polymer expansions of statistical mechanics. Despite the fact that our approach is elementary, our results are slightly sharper than those contained in the literature up to now. We show that, whenever the small quantum system is known to admit a Markov approximation (Pauli master equation \emph{aka} Lindblad equation) in the weak coupling limit, and the Markov approximation is exponentially mixing, then the weakly coupled system approaches a unique invariant state that is perturbatively close to its Markov approximation.Comment: 23 pages, v2-->v3: Revised version: The explanatory section 1.7 has changed and Section 3.2 has been made more explici

Non-commutative Geometry and Kinetic Theory of Open Systems

The basic mathematical assumptions for autonomous linear kinetic equations for a classical system are formulated, leading to the conclusion that if they are differential equations on its phase space $M$, they are at most of the 2nd order. For open systems interacting with a bath at canonical equilibrium they have a particular form of an equation of a generalized Fokker-Planck type. We show that it is possible to obtain them as Liouville equations of Hamiltonian dynamics on $M$ with a particular non-commutative differential structure, provided certain geometric in character, conditions are fulfilled. To this end, symplectic geometry on $M$ is developped in this context, and an outline of the required tensor analysis and differential geometry is given. Certain questions for the possible mathematical interpretation of this structure are also discussed.Comment: 22 pages, LaTe

Open Quantum Dynamics: Complete Positivity and Entanglement

We review the standard treatment of open quantum systems in relation to quantum entanglement, analyzing, in particular, the behaviour of bipartite systems immersed in a same environment. We first focus upon the notion of complete positivity, a physically motivated algebraic constraint on the quantum dynamics, in relation to quantum entanglement, i.e. the existence of statistical correlations which can not be accounted for by classical probability. We then study the entanglement power of heat baths versus their decohering properties, a topic of increasing importance in the framework of the fast developing fields of quantum information, communication and computation. The presentation is self contained and, through several examples, it offers a detailed survey of the physics and of the most relevant and used techniques relative to both quantum open system dynamics and quantum entanglement.Comment: LaTex, 77 page

Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast

To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non‐perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half‐lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress‐responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress‐induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. DTA‐derived mRNA synthesis rates identified 16 stress‐specific pairs/triples of cooperative transcription factors, of which seven were known. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems