Detecting Mutations in the Mycobacterium tuberculosis Pyrazinamidase Gene pncA to Improve Infection Control and Decrease Drug Resistance Rates in Human Immunodeficiency Virus Coinfection.

Hospital infection control measures are crucial to tuberculosis (TB) control strategies within settings caring for human immunodeficiency virus (HIV)-positive patients, as these patients are at heightened risk of developing TB. Pyrazinamide (PZA) is a potent drug that effectively sterilizes persistent Mycobacterium tuberculosis bacilli. However, PZA resistance associated with mutations in the nicotinamidase/pyrazinamidase coding gene, pncA, is increasing. A total of 794 patient isolates obtained from four sites in Lima, Peru, underwent spoligotyping and drug resistance testing. In one of these sites, the HIV unit of Hospital Dos de Mayo (HDM), an isolation ward for HIV/TB coinfected patients opened during the study as an infection control intervention: circulating genotypes and drug resistance pre- and postintervention were compared. All other sites cared for HIV-negative outpatients: genotypes and drug resistance rates from these sites were compared with those from HDM. HDM patients showed high concordance between multidrug resistance, PZA resistance according to the Wayne method, the two most common genotypes (spoligotype international type [SIT] 42 of the Latino American-Mediterranean (LAM)-9 clade and SIT 53 of the T1 clade), and the two most common pncA mutations (G145A and A403C). These associations were absent among community isolates. The infection control intervention was associated with 58-92% reductions in TB caused by SIT 42 or SIT 53 genotypes (odds ratio [OR] = 0.420, P = 0.003); multidrug-resistant TB (OR = 0.349, P < 0.001); and PZA-resistant TB (OR = 0.076, P < 0.001). In conclusion, pncA mutation typing, with resistance testing and spoligotyping, was useful in identifying a nosocomial TB outbreak and demonstrating its resolution after implementation of infection control measures

Vertical flow in the Southern Ocean estimated from individual moorings

In this study, we demonstrate that oceanic vertical velocities can be estimated from individual mooring measurements, even for non-stationary flow. This result is obtained under three assumptions: i. weak diffusion (Péclet number ≫1), ii. weak friction (Reynolds number ≫1), and iii. small inertial terms (Rossby number ≪1). The theoretical framework is applied to a set of 4 moorings located in the Southern Ocean. For this site, the diagnosed vertical velocities are highly variable in time, their standard deviation being one-to-two orders of magnitude greater than their mean. We demonstrate that the time-averaged vertical velocities are largely induced by geostrophic flow, and can be estimated from the time-averaged density and horizontal velocities. This suggests that local time-mean vertical velocities are primarily forced by the time-mean ocean dynamics, rather than by e.g. transient eddies or internal waves. We also show that, in the context of these four moorings, the time-mean vertical flow is consistent with stratified Taylor column dynamics in the presence of a topographic obstacle

Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity.

Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT-Apo and PZAse-EC-Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 μM) achieved 65% PZAse-EC-Apo reactivation. Rv2059 (1 μM) and ZnuA (1 μM) achieved 69% and 34.3% PZAse-MT-Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT-Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAse in vitro Although Rv2059 is important in vitro, it seems to have a smaller effect on PZA susceptibility in vivo. It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis Further studies are needed for confirmation.IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide's mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient's individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat

A programme for the prevention of post-traumatic stress disorder in midwifery (POPPY): indications of effectiveness from a feasibility study

Background: Midwives can experience events they perceive as traumatic when providingcare. As a result, some will develop post-traumatic stress disorder (PTSD), with adverse implications for their mental health, the quality of care provided for women and the employing organizations. POPPY (Programme for the prevention of PTSD in midwifery) is a package of educational and supportive resources comprising an educational workshop, information leaflet, peer support and access to trauma-focused clinical psychology intervention. A feasibility study of POPPY implementation was completed. Objective: This study aimed to identify potential impacts of POPPY on midwives’ understandingof trauma, their psychological well-being and job satisfaction. Method: POPPY was implemented in one hospital site. Before taking part in the POPPY workshop (T1) midwives (N = 153) completed self-report questionnaires, which measured exposure to work-related trauma, knowledge and confidence of managing trauma responses, professional impacts, symptoms of PTSD, burnout and job satisfaction. Measures were repeated (T2) approximately 6 months after training (n = 91, 62%). Results: Midwives’ confidence in recognizing (p = .001) and managing early traumaresponses in themselves and their colleagues significantly improved (both p < .001). There was a trend towards reduced levels of PTSD symptomatology, and fewer midwives reported sub clinical levels of PTSD (from 10% at T1 to 7% at T2). The proportion of midwives reporting high and moderate levels of depersonalization towards care was reduced (33% to 20%) and midwives reported significantly higher levels of job satisfaction at T2 (p < .001). Reductions in self-reported stress-related absenteeism (12% to 5%), long-term changes to clinical allocation (10% to 5%) and considerations about leaving midwifery (34% to 27%) were identified. Conclusions: In conclusion, POPPY shows very positive potential to improve midwives’ mental health and the sensitivity of care they provide, and reduce service disruption and costs for trusts. Large-scale longitudinal evaluation is required

GASP II. A MUSE view of extreme ram-pressure stripping along the line of sight: kinematics of the jellyfish galaxy JO201

This paper presents a spatially-resolved kinematic study of the jellyfish galaxy JO201, one of the most spectacular cases of ram-pressure stripping (RPS) in the GASP (GAs Stripping Phenomena in Galaxies with MUSE) survey. By studying the environment of JO201, we find that it is moving through the dense intra-cluster medium of Abell 85 at supersonic speeds along our line of sight, and that it is likely accompanied by a small group of galaxies. Given the density of the intra-cluster medium and the galaxy's mass, projected position and velocity within the cluster, we estimate that JO201 must so far have lost ~50% of its gas during infall via RPS. The MUSE data indeed reveal a smooth stellar disk, accompanied by large projected tails of ionised (Halpha) gas, composed of kinematically cold (velocity dispersion <40km/s) star-forming knots and very warm (>100km/s) diffuse emission which extend out to at least ~50 kpc from the galaxy centre. The ionised Halpha-emitting gas in the disk rotates with the stars out to ~6 kpc but in the disk outskirts becomes increasingly redshifted with respect to the (undisturbed) stellar disk. The observed disturbances are consistent with the presence of gas trailing behind the stellar component, resulting from intense face-on RPS happening along the line of sight. Our kinematic analysis is consistent with the estimated fraction of lost gas, and reveals that stripping of the disk happens outside-in, causing shock heating and gas compression in the stripped tails.Comment: ApJ, revised version after referee comments, 15 pages, 16 figures. The interactive version of Figure 9 can be viewed at web.oapd.inaf.it/gasp/publications.htm

Modification of turbulent dissipation rates by a deep Southern Ocean eddy

The impact of a mesoscale eddy on the magnitude and spatial distribution of diapycnal ocean mixing is investigated using a set of hydrographic and microstructure measurements collected in the Southern Ocean. These data sampled a baroclinic, mid-depth eddy formed during the disintegration of a deep boundary current. Turbulent dissipation is suppressed within the eddy, but is elevated by up to an order of magnitude along the upper and lower eddy boundaries. A ray-tracing approximation is employed asa heuristic device to elucidate how the internal wave field evolves in the ambient velocity and stratification conditions accompanying the eddy. These calculations are consistent with the observations, suggesting reflection of internal wave energy from the eddy center and enhanced breaking through critical layer processes along the eddy boundaries. These results have important implications for understanding where and how internal wave energy is dissipated in the presence of energetic deep geostrophic flows

The detection of airborne transmission of tuberculosis from HIV-infected patients, using an in vivo air sampling model

Background. Nosocomial transmission of tuberculosis remains an important public health problem. We created an in vivo air sampling model to study airborne transmission of tuberculosis from patients coinfected with human immunodeficiency virus (HIV) and to evaluate environmental control measures. Methods. An animal facility was built above a mechanically ventilated HIV‐tuberculosis ward in Lima, Peru. A mean of 92 guinea pigs were continuously exposed to all ward exhaust air for 16 months. Animals had tuberculin skin tests performed at monthly intervals, and those with positive reactions were removed for autopsy and culture for tuberculosis. Results. Over 505 consecutive days, there were 118 ward admissions by 97 patients with pulmonary tuberculosis, with a median duration of hospitalization of 11 days. All patients were infected with HIV and constituted a heterogeneous group with both new and existing diagnoses of tuberculosis. There was a wide variation in monthly rates of guinea pigs developing positive tuberculin test results (0%–53%). Of 292 animals exposed to ward air, 159 developed positive tuberculin skin test results, of which 129 had laboratory confirmation of tuberculosis. The HIV‐positive patients with pulmonary tuberculosis produced a mean of 8.2 infectious quanta per hour, compared with 1.25 for HIV‐negative patients with tuberculosis in similar studies from the 1950s. The mean monthly patient infectiousness varied greatly, from production of 0–44 infectious quanta per hour, as did the theoretical risk for a health care worker to acquire tuberculosis by breathing ward air. Conclusions. HIV‐positive patients with tuberculosis varied greatly in their infectiousness, and some were highly infectious. Use of environmental control strategies for nosocomial tuberculosis is therefore a priority, especially in areas with a high prevalence of both tuberculosis and HIV infection

Supporting researchers conducting qualitative research into sensitive, challenging, and difficult topics: Experiences and practical applications.

Qualitative researchers often engage in work addressing challenging, difficult, or sensitive topics and are consequently exposed to the participants’ narratives which may be emotionally charged, distressing, or compromising. These narratives occasionally rest heavy on a researcher’s conscience or may linger in the mind. Much literature has assessed how best to keep participants safe, but less attention has been given to how we keep researchers safe. We therefore document the following: (1) Our experiences of the issues presented by undertaking qualitative research involving challenging, difficult, or sensitive topics; and (2) Practical principles devised to overcome these issues, ensuring safety and wellbeing amongst researchers engaging in these types of qualitative research. We provide guidance for qualitative researchers of all levels of experience and expertise on how best to protect and support themselves, their colleagues, and other collaborating research staff, when undertaking qualitative research which might otherwise feel uncomfortable or overwhelming to tackle

Proteome Profiling of Breast Tumors by Gel Electrophoresis and Nanoscale Electrospray Ionization Mass Spectrometry

We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future. © 2008 American Chemical Society

Genetic diversity of Mycobacterium tuberculosis in Peru and exploration of phylogenetic associations with drug resistance.

BACKGROUND: There is limited available data on the strain diversity of M tuberculosis in Peru, though there may be interesting lessons to learn from a setting where multidrug resistant TB has emerged as a major problem despite an apparently well-functioning DOTS control programme. METHODS: Spoligotyping was undertaken on 794 strains of M tuberculosis collected between 1999 and 2005 from 553 community-based patients and 241 hospital-based HIV co-infected patients with pulmonary tuberculosis in Lima, Peru. Phylogenetic and epidemiologic analyses permitted identification of clusters and exploration of spoligotype associations with drug resistance. RESULTS: Mean patient age was 31.9 years, 63% were male and 30.4% were known to be HIV+. Rifampicin mono-resistance, isoniazid mono-resistance and multidrug resistance (MDR) were identified in 4.7%, 8.7% and 17.3% of strains respectively. Of 794 strains from 794 patients there were 149 different spoligotypes. Of these there were 27 strains (3.4%) with novel, unique orphan spoligotypes. 498 strains (62.7%) were clustered in the nine most common spoligotypes: 16.4% SIT 50 (clade H3), 12.3% SIT 53 (clade T1), 8.3% SIT 33 (LAM3), 7.4% SIT 42 (LAM9), 5.5% SIT 1 (Beijing), 3.9% SIT 47 (H1), 3.0% SIT 222 (clade unknown), 3.0% SIT1355 (LAM), and 2.8% SIT 92 (X3). Amongst HIV-negative community-based TB patients no associations were seen between drug resistance and specific spoligotypes; in contrast HIV-associated MDRTB, but not isoniazid or rifampicin mono-resistance, was associated with SIT42 and SIT53 strains. CONCLUSION: Two spoligotypes were associated with MDR particularly amongst patients with HIV. The MDR-HIV association was significantly reduced after controlling for SIT42 and SIT53 status; residual confounding may explain the remaining apparent association. These data are suggestive of a prolonged, clonal, hospital-based outbreak of MDR disease amongst HIV patients but do not support a hypothesis of strain-specific propensity for the acquisition of resistance-conferring mutations