Role and value of whole genome sequencing in studying tuberculosis transmission

BackgroundTuberculosis (TB) remains a serious public health threat worldwide. Theoretically ultimate resolution of whole genome sequencing (WGS) for Mycobacterium tuberculosis complex (MTBC) strain classification makes this technology very attractive for epidemiological investigations.ObjectivesTo summarize the evidence available in peer-reviewed publications on the role and place of WGS in detection of TB transmission.SourcesA total of 69 peer-reviewed publications identified in Pubmed database.ContentEvidence from >30 publications suggests that a cut-off value of fewer than six single nucleotide polymorphisms between strains efficiently excludes cases that are not the result of recent transmission and could be used for the identification of drug-sensitive isolates involved in direct human-to-human TB transmission. Sensitivity of WGS to identify epidemiologically linked isolates is high, reaching 100% in eight studies with specificity (17%–95%) highly dependent on the settings. Drug resistance and specific phylogenetic lineages may be associated with accelerated mutation rates affecting genetic distances. WGS can be potentially used to distinguish between true relapses and re-infections but in high-incidence low-diversity settings this would require consideration of epidemiological links and minority alleles. Data from four studies looking into within-host diversity highlight a need for developing criteria for acceptance or rejection of WGS relatedness results depending on the proportion of minority alleles.ImplicationsWGS will potentially allow for more targeted public health actions preventing unnecessary investigations of false clusters. Consensus on standardization of raw data quality control processing criteria, analytical pipelines and reporting language is yet to be reached.     

Evaluation of two molecular assays for rapid detection of mycobacterium tuberculosis resistance to fluoroquinolones in high-tuberculosis and -multidrug-resistance Settings

The Russian Federation is a high-tuberculosis (TB)-burden country with high rates of drug resistance, including multidrug and extensive drug resistance to TB (M/XDRTB). Rapid diagnosis of resistance to fluoroquinolones (FQs) using molecular assays is essential for the implementation of appropriate drug regimens and prevention of the transmission of XDR strains. A total of 51 individual MDRTB strains were tested by pyrosequencing of the quinolone resistance determining region of the gyrA gene and the GenoType MTBDRsl assay (Hain Lifescience, GmbH, Nehren, Germany), and the results were evaluated against those obtained by phenotypic drug susceptibility testing (DST). Mutations were detected in 25 (78.1%) FQ-resistant strains, with the majority of mutations (n = 19 [73.0%]) found in codon 94 of the gyrA gene; the novel mutation 1457 C→Τ was found in the gyrB gene. Three mixed allelic variants were detected, which is a well-known phenomenon in areas with high TB and drug-resistant TB rates. The sensitivity and specificity of pyrosequencing (86.2 and 100%, respectively) and MTBDRsl (86.2 and 100%, respectively) were high; however, the results for 5.9% of the analyzed strains were unreadable when MTBDRsl was used. The MTBDRsl and pyrosequencing assays offer a rapid and accurate means for diagnosing resistance to FQs in high-TB-burden areas.     

Detection of resistance to second-line antituberculosis drugs by use of the genotype MTBDRsl assay: a multicenter evaluation and feasibility study

The rate of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has been steadily increasing in countries of the former USSR. The availability of rapid and reliable methods for the detection of drug resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the Genotype MTBDRsl assay compared to that of phenotypic drug susceptibility testing (Becton Dickinson Bactec MGIT 960 system) with a test panel of 200 Mycobacterium tuberculosis isolates at four sites in Eastern Europe. The interpretability of the Genotype MTBDRsl assay was over 95%. The sensitivity for the detection of resistance to fluoroquinolones, ethambutol, amikacin, and capreomycin varied between 77.3% and 92.3%; however, it was much lower for kanamycin (42.7%). The sensitivity for the detection of XDR TB was 22.6%. The test specificity was over 82% for all drugs. The assay presents a good screening tool for the rapid detection of resistance to individual second-line drugs and can be recommended for use in countries with a high burden of MDR/XDR TB. The sensitivity for the detection of kanamycin resistance needs improvement.     

Associations between Mycobacterium tuberculosis Strains and Phenotypes

To inform development of tuberculosis (TB) control strategies, we characterized a total of 2,261 Mycobacterium tuberculosis complex isolates by using multiple phenotypic and molecular markers, including polymorphisms in repetitive sequences (spoligotyping and variable-number tandem repeats [VNTRs]) and large sequence and single-nucleotide polymorphisms. The Beijing family was strongly associated with multidrug resistance (p = 0.0001), and VNTR allelic variants showed strong associations with spoligotyping families: >5 copies at exact tandem repeat (ETR) A, >2 at mycobacterial interspersed repetitive unit 24, and >3 at ETR-B associated with the East African–Indian and M. bovis strains. All M. tuberculosis isolates were differentiated into 4 major lineages, and a maximum parsimony tree was constructed suggesting a more complex phylogeny for M. africanum. These findings can be used as a model of pathogen global diversity.     

The relationship between fasting-induced torpor,sleep, and wakefulness in laboratory mice

Study ObjectivesTorpor is a regulated and reversible state of metabolic suppression used by many mammalian species to conserve energy. Whereas the relationship between torpor and sleep has been well-studied in seasonal hibernators, less is known about the effects of fasting-induced torpor on states of vigilance and brain activity in laboratory mice.MethodsContinuous monitoring of electroencephalogram (EEG), electromyogram (EMG), and surface body temperature was undertaken in adult, male C57BL/6 mice over consecutive days of scheduled restricted feeding.ResultsAll animals showed bouts of hypothermia that became progressively deeper and longer as fasting progressed. EEG and EMG were markedly affected by hypothermia, although the typical electrophysiological signatures of non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep, and wakefulness enabled us to perform vigilance-state classification in all cases. Consistent with previous studies, hypothermic bouts were initiated from a state indistinguishable from NREM sleep, with EEG power decreasing gradually in parallel with decreasing surface body temperature. During deep hypothermia, REM sleep was largely abolished, and we observed shivering-associated intense bursts of muscle activity.ConclusionsOur study highlights important similarities between EEG signatures of fasting-induced torpor in mice, daily torpor in Djungarian hamsters and hibernation in seasonally hibernating species. Future studies are necessary to clarify the effects on fasting-induced torpor on subsequent sleep.     

Re-examining extreme sleep duration in bats: implications for sleep phylogeny,ecology, and function

Bats, quoted as sleeping for up to 20 h a day, are an often used example of extreme sleep duration amongst mammals. Given that duration has historically been one of the primary metrics featured in comparative studies of sleep, it is important that species specific sleep durations are well founded. Here, we re-examined the evidence for the characterization of bats as extreme sleepers and discuss whether it provides a useful representation of the sleep behavior of Chiroptera. Although there are a wealth of activity data to suggest that the diurnal cycle of bats is dominated by rest, estimates of sleep time generated from electrophysiological analyses suggest considerable interspecific variation, ranging from 83% to a more moderate 61% of the 24 h day spent asleep. Temperature-dependent changes in the duration and electroencephalographic profile of sleep suggest that bats represent a unique model for investigating the relationship between sleep and torpor. Further sources of intra-specific variation in sleep duration, including the impact of artificial laboratory environments and sleep intensity, remain unexplored. Future studies conducted in naturalistic environments, using larger sample sizes and relying on a pre-determined set of defining criteria will undoubtedly provide novel insights into sleep in bats and other species.     

Computer Modelling of Energy Structure of Yb3+ and Lu3+ Doped LaF3 Crystals

The energy band structure, as well as partial and total densities of states have been calculated for LaF₃:Yb and LaF₃:Lu crystals within density functional theory using the projector augmented wave method and Hubbard corrections (DFT + U). The influence of geometric optimization on the results of energy band calculations of LaF₃:Ln crystals (Ln = Yb, Lu) was analysed and the absence of relaxation procedure is confirmed to negatively influence the energy position of states, and the variability between obtained results of different optimization algorithms are within the calculation accuracy. The top of the valence band of LaF₃ is confirmed to be formed by the 2pF^--states and the bottom of the conduction band is formed by the 5d-states of La³⁺. The positions of the 4f-states and 5d-states of activator ions in LaF₃ were studied. It is shown that the 4f-states of Yb³⁺ are slightly above the top of the valence band and the 4f-states of Lu³⁺ to be 3.5 eV below the top of the valence band. The energy levels of the 5d states of the impurities are energetically close to the bottom of the LaF₃ conduction band. The calculated band gap of 9.6 eV for LaF₃ is in a good agreement with the experimental result and is not affected by impurity ions.