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The PhoPR system is a master regulator in Mycobacterium tuberculosis. A key difference between M. tuberculosis and Mycobacterium bovis is a G71I substitution in the M. bovis PhoR orthologue. Functional studies of the M. bovis PhoPR system have generated conflicting findings, with some research suggesting that the M. bovis PhoPR is defective while others indicate it is functional. We sought to revisit the functionality of the M. bovis PhoPR system. To address this, we constructed a phoPR mutant in the reference strain M. bovis AF2122/97. We employed a combination of growth assays and transcriptomics analyses to assess the phenotype of the mutant vs wild type and complemented strains. We found that the M. bovis AF2122/97 Δ phoPR mutant showed a growth defect on solid and liquid media compared to the wild type and complemented strains. The transcriptome of the M. bovis AF2122/97 Δ phoPR mutant was also altered as compared to wild type, including differential expression of genes involved in lipid metabolism and secretion. Our work provides further insight into the activity of PhoPR in M. bovis and underlines the importance of the PhoPR system as a master regulator of gene expression in the Mycobacterium tuberculosis complex. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis , the causative agent of human tuberculosis (hTB), is a close evolutionary relative of Mycobacterium bovis , which causes bovine tuberculosis (bTB), one of the most damaging infectious diseases to livestock agriculture. Previous studies have shown that the pathogenesis of bTB disease is comparable to hTB disease, and that the bovine and human alveolar macrophage (bAM and hAM, respectively) transcriptomes are extensively reprogrammed in response to infection with these intracellular mycobacterial pathogens. In this study, a multi-omics integrative approach was applied with functional genomics and GWAS data sets across the two primary hosts (Bos taurus and Homo sapiens) and both pathogens (M. bovis and M. tuberculosis). Four different experimental infection groups were used: 1) bAM infected with M. bovis , 2) bAM infected with M. tuberculosis , 3) hAM infected with M. tuberculosis , and 4) human monocyte-derived macrophages (hMDM) infected with M. tuberculosis. RNA-seq data from these experiments 24 h post-infection (24 hpi) was analysed using three computational pipelines: 1) differentially expressed genes, 2) differential gene expression interaction networks, and 3) combined pathway analysis. The results were integrated with high-resolution bovine and human GWAS data sets to detect novel quantitative trait loci (QTLs) for resistance to mycobacterial infection and resilience to disease. This revealed common and unique response macrophage pathways for both pathogens and identified 32 genes (12 bovine and 20 human) significantly enriched for SNPs associated with disease resistance, the majority of which encode key components of the NF-κB signalling pathway and that also drive formation of the granuloma. [ABSTRACT FROM AUTHOR]

Primary amputation (ie, without attempted revascularization) is a devastating complication of peripheral artery disease. Racial disparities in primary amputation have been described; however, rural disparities have not been well investigated. The purpose of this study was to examine the impact of rurality on risk of primary amputation and to explore the effect of race on this relationship. The national Vascular Quality Initiative amputation data set was used for analyses (N = 6795). The outcome of interest was primary amputation. Independent variables were race/ethnicity (non-Latinx whites vs nonwhites) and rural residence. Multivariable logistic regression examined impact of rurality and race/ethnicity on primary amputation after adjustment for relevant covariates and included an interaction for race/ethnicity by rural status. Primary amputation occurred in 49% of patients overall (n = 3332), in 47% of rural vs 49% of urban patients (P =.322), and in 46% of whites vs 53% of nonwhites (P <.001). On multivariable analysis, nonwhites had a 21% higher odds of undergoing primary amputation overall (adjusted odds ratio [AOR], 1.21; 95% confidence interval [CI], 1.05-1.39). On subgroup analysis, rural nonwhites had two times higher odds of undergoing primary amputation than rural whites (AOR, 2.06; 95% CI, 1.53-2.78) and a 52% higher odds of undergoing primary amputation than urban nonwhites (AOR, 1.52; 95% CI, 1.19-1.94). In the urban setting, nonwhites had a 21% higher odds of undergoing primary amputation than urban whites (AOR, 1.21; 95% CI, 1.05-1.39). In these analyses, rurality was associated with greater odds for primary amputation in nonwhite patients but not in white patients. The effect of race on primary amputation was significant in both urban and rural settings; however, the effect was significantly stronger in rural settings. These findings suggest that race/ethnicity has a compounding effect on rural health disparities and that strategies to improve health of rural communities need to consider the particular needs of nonwhite residents to reduce disparities. [ABSTRACT FROM AUTHOR]

The purpose of this study was to develop a simple visual methyl green (MeG) based dry loop-mediated isothermal amplification (LAMP) method for early detection of Mycobacterium tuberculosis (MTB) from clinical samples. We identified MeG as an indicator of a positive LAMP reaction, where a positive reaction gave a blue-green color while a negative reaction was colorless. The MeG MTB-LAMP system was further simplified by drying all reagents for ease of use, and was then validated for its ability to diagnose TB directly using Nepalese clinical samples. We evaluated the dry MeG MTB-LAMP with 69 new TB suspected samples from patients that did not have a confirmed history of TB treatment and found the sensitivity in culture positive samples as 92.8% (13/14) and specificity in culture negative samples as 96.3% (53/55). Our LAMP system has the potential to be a point of care test for early diagnosis of active TB in developing countries. [ABSTRACT FROM AUTHOR]

Objectives: We aimed to describe the analgesic efficacy, duration of analgesia, and adverse event profile associated with intranasal hydromorphone in children with acute pain presenting to an emergency department.Methods: Prospective dose titration pilot study of otherwise healthy children 4 to 17-years-old with moderate to severe pain who required a parenteral opioid. All patients received an initial intranasal hydromorophone dose of 0.03 mg/kg. The need for additional analgesia was assessed at 15 and 30 min; an additional 0.015 mg/kg was given at each assessment, if required. Need for rescue analgesic, pain intensity and adverse events were assessed until 6 h after hydromorphone administration or until patients were discharged, underwent a procedure to treat their painful condition, or received a rescue analgesic.Results: We enrolled 35 children. Fifteen, 11, and 9 children required a total dose of 0.03, 0.045, and 0.06 mg/kg, respectively. Patients in each dose group experienced an absolute decrease in pain score of ≥3/10 and percent reduction >40% within 5-15 min of completing dose-titration administration of hydromorphone. Duration of analgesia (i.e. time until rescue analgesic administered) >1 h was observed in 85.7% of patients. Patients not requiring rescue analgesics had mild or no pain until discharged or their painful conditions were treated. Three (8.6%) patients required a rescue analgesic <1 h after hydromorphone administration. There were no major adverse events.Conclusions: Intranasal hydromorphone led to rapid, clinically significant and frequently sustained decreases in pain intensity in children. No major adverse events were observed in this preliminary sample. Clinical Trials Registration Number: NCT02437669. [ABSTRACT FROM AUTHOR]

Abstract Clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis are differentially susceptible to 2-Thiophen Hydrazide (TCH); however its mechanism of action or the reasons for that difference are unknown. We report herein that under our experimental conditions, TCH inhibits M. tuberculosis in solid but not in liquid medium, and that in spite of resembling Isoniazid and Ethionamide, it does not affect mycolic acid synthesis. To understand the mechanisms of action of TCH we isolated M. tuberculosis TCH resistant mutants which fell into two groups; one resistant to TCH and Isoniazid but not to Ethionamide or Triclosan, and the other resistant only to TCH with no, or marginal, cross resistance to Isoniazid. A S315T katG mutation conferred resistance to TCH while kat G expression from a plasmid reduced M. tuberculosis MIC to this drug, suggesting a possible involvement of KatG in TCH activation. Whole genome sequencing of mutants from this second group revealed a single mutation in the alkylhydroperoxide reductase ahp C promoter locus in half of the mutants, while the remaining contained mutations in dispensable genes. This is the first report of the genetics underlying the action of TCH and of the involvement of ahpC as the sole basis for resistance to an anti-tubercular compound. [ABSTRACT FROM AUTHOR]

Summary Coordinated regulation of gene expression is essential for pathogen adaptation in vivo . Understanding the control of these virulence circuits in the TB pathogen Mycobacterium tuberculosis is a key challenge if we are to increase our basic understanding of how this organism establishes infection. In this study we focused on the transcriptional regulator Rv1404 that shows similarity to the MarR family of transcriptional repressors. Rv1404 derepresses a set of genes in vivo that have been implicated in virulence and may therefore allow adaptation of M. tuberculosis to the intracellular environment. We used a combination of ChIP-qPCR and Electromobility Band Shift Assays (EMSA) to show that Rv1404 coordinates gene expression in response to stresses such as low pH in M. tuberculosis . Two genes regulated by Rv1404, rv1403c and rv1405c , encode putative SAM-dependent methyltransferases. To elucidate gene function, M. tuberculosis rv1403c and rv1405c mutants were constructed. The mutants showed attenuated growth in response to in vitro stress conditions that mimic the intracellular milieu. Our data sheds new light on the function of a novel regulon controlled by Rv1404 that coordinates adaptation of M. tuberculosis to the in vivo environment and reveals the Rv1405c and Rv1403c methyltransferases as playing a role in this adaptive process. [ABSTRACT FROM AUTHOR]

Improved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response of Mycobacterium bovis -infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood—an important component of current bTB diagnostics—will provide new information for development of better diagnostics. RNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (−1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-week M. bovis infection time course experiment with ten age-matched Holstein-Friesian cattle. In vitro PPD-b stimulation of peripheral blood from M. bovis -infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters. A transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers of M. bovis infection. [ABSTRACT FROM AUTHOR]

Summary Mycobacterium bovis , the causative agent of bovine tuberculosis, a major problem for global agriculture, spreads via an airborne route and is taken up by alveolar macrophages (AM) in the lung. Here, we describe the first next-generation sequencing (RNA-seq) approach to temporally profile miRNA expression in primary bovine AMs post-infection with M . bovis . One, six, and forty miRNAs were identified as significantly differentially expressed at 2, 24 and 48 h post-infection, respectively. The differential expression of three miRNAs (bta-miR-142-5p, bta-miR-146a, and bta-miR-423-3p) was confirmed by RT-qPCR. Pathway analysis of the predicted mRNA targets of differentially expressed miRNAs suggests that these miRNAs preferentially target several pathways that are functionally relevant for mycobacterial pathogenesis, including endocytosis and lysosome trafficking, IL-1 signalling and the TGF-β pathway. Over-expression studies using a bovine macrophage cell-line (Bomac) reveal the targeting of two key genes in the innate immune response to M. bovis , IL-1 receptor-associated kinase 1 (IRAK1) and TGF-β receptor 2 (TGFBR2) , by miR-146. Taken together, our study suggests that miRNAs play a key role in tuning the complex interplay between M. bovis survival strategies and the host immune response. [ABSTRACT FROM AUTHOR]

Summary Objective Bacterial meningitis in sub-Saharan Africa is predominantly caused by Streptococcus pneumoniae , is often associated with HIV co-infection and mortality rates are double those seen in better resourced settings. Methods To investigate the cause of this excessive mortality we quantified the pneumococcal DNA load and six common pro-inflammatory cytokines in the cerebrospinal fluid (CSF) of Malawian adults with culture proven pneumococcal meningitis and correlated the results to clinical parameters and outcome. There are currently no published data relating bacterial load to outcome in adults with pneumococcal meningitis. Results The mean age of patients was 32 years, 82% were HIV infected and 49% had died by day 40. CSF bacterial loads were high (median 6.5 × 10 5 copies/ml CSF) and there was no significant variation in bacterial load between survivors and non-survivors. All pro-inflammatory CSF cytokines were elevated in the CSF, with no clinically important differences between survivors and non-survivors. HIV status did not affect the CSF bacterial load or cytokine response. Conclusion Mortality from pneumococcal meningitis in adults in sub-Saharan Africa is not related to pneumococcal bacterial load. More research is needed to understand the very high mortality from meningitis in this region. [ABSTRACT FROM AUTHOR]

Summary: Despite sharing >99.9% genome sequence similarity at the nucleotide level, Mycobacterium tuberculosis and Mycobacterium bovis—the causative agents of human and bovine tuberculosis, respectively—exhibit distinct host preferences. M. bovis can cause disease in both cattle and humans yet rarely transmits between immuno-competent human hosts, while M. tuberculosis is a highly successful pathogen of humans that does not sustain in animal populations. Based on the key role played by alveolar macrophages during mycobacterial infection, we hypothesised that the immunological and pathological differences observed in cattle infected with virulent M. bovis and M. tuberculosis may have a basis in innate immune mechanisms; these differences, in turn, would be reflected at the macrophage mRNA and protein level. To investigate this, we have analysed the transcriptional profile of innate immune genes in bovine alveolar macrophages following 24 and 48 h infection with the genome-sequenced strains, M. bovis AF2122/97 and M. tuberculosis H37Rv. A bespoke multiplex ELISA was also used to quantify corresponding cytokine secretion in supernatants from the same infected alveolar macrophages. All cytokines showed similar significant patterns of expression (i.e. up- or down-regulation) at both the mRNA and protein levels in infected macrophages relative to parallel non-infected controls at the two time points (P ≤ 0.05). However, significant upregulation and downregulation of several innate immune genes—including TLR2, FOS, PIK3IP1, CCL4, IL1B, IL6 and TNF—and the CCL-4 protein was observed in the M. bovis-infected macrophages relative to the M. tuberculosis-infected macrophages 48 h post-infection (P ≤ 0.05). These results support the hypothesis that the divergent virulence of M. bovis and M. tuberculosis in cattle has a basis in innate immune mechanisms, which may contribute to host preference within the M. tuberculosis complex of strains. [Copyright &y& Elsevier]

Summary: Access to online repositories for genomic and associated “-omics” datasets is now an essential part of everyday research activity. It is important therefore that the Tuberculosis community is aware of the databases and tools available to them online, as well as for the database hosts to know what the needs of the research community are. One of the goals of the Tuberculosis Annotation Jamboree, held in Washington DC on March 7th–8th 2012, was therefore to provide an overview of the current status of three key Tuberculosis resources, TubercuList (tuberculist.epfl.ch), TB Database (www.tbdb.org), and Pathosystems Resource Integration Center (PATRIC, www.patricbrc.org). Here we summarize some key updates and upcoming features in TubercuList, and provide an overview of the PATRIC site and its online tools for pathogen RNA-Seq analysis. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis, caused by Mycobacterium bovis (M. bovis), is a globally distributed chronic disease of animals. The bacteria can be transmitted to humans via the consumption of unpasteurised (raw) milk, thus representing an important public health risk. To investigate the risk of zoonotic transmission of M. bovis via raw milk, this study systematically reviewed published studies to estimate the prevalence of M. bovis in on-farm bulk-tank milk (BTM) and individual cow's milk (IM) by meta-analysis. In total, 1,339 articles were identified through seven electronic databases and initially screened using titles and abstracts. The quality of 108 potentially relevant articles was assessed using full texts, and 67 articles comprising 83 studies (76 IM and 7 BTM), were included in the meta-analysis. The prevalence of M. bovis in IM and BTM was summarised according to the diagnostic test used, and the tuberculin skin test (TST) infection status of the individual cows (for IM) or herds (for BTM). Heterogeneity was quantified using the I -squared statistic. Prediction intervals (95% PIs) were also estimated. For IM, the overall prevalence was summarised at 5% (95%CI: 3%–7%). In TST positive cows, prevalence was summarised at 8% (95%CI: 4%–13%). For BTM, the overall prevalence independent of individual herd TST infection status was summarised at 5% (95%CI: 0%–21%). There was considerable heterogeneity evident among the included studies, while PIs were also wide. Inconsistency in the quality of reporting was also observed resulting in missing information, such as the TST infection status of the individual animal/herd. No study reported the number of M. bovis bacteria in test-positive milk samples. Several studies reported the detection of M. tuberculosis and M. africanum in milk. Despite international efforts to control tuberculosis, this study highlights the risk of zoonotic transmission of M. bovis via unpasteurised milk and dairy products made using raw milk. [ABSTRACT FROM AUTHOR]

Reports on a study on the pharmacokinetics of a seven-day estradiol transdermal system (Climara) to investigate the efficacy, safety and wearability of estrogen replacement therapy using a patch delivery system on menopausal women. Description of the 7-day transdermal system; Results showing that the patch is well tolerated and effective.

Amputation is a devastating but preventable complication of diabetes and peripheral arterial disease (PAD). Multiple studies have focused on disparities in amputation rates based on race and socioeconomic status, but few focus on amputation trends in rural populations. The objective of this study was to identify the prevalence of major and minor amputation among patients admitted with diabetes and/or PAD in a rural, Appalachian state, and to identify geographic areas with higher than expected major and minor amputations using advanced spatial analysis while controlling for comorbidities and rurality. Patient hospital admissions of West Virginia residents with diagnoses of diabetes and/or PAD and with or without an amputation procedure were identified from the West Virginia Health Care Authority State Inpatient Database from 2011 to 2016 using relevant International Classification of Diseases , 9th edition and 10the edition codes. Bayesian spatial hierarchical modeling was conducted to identify areas of high risk, while controlling for important confounders for amputation. Overall, there were 5557 amputations among 459,452 hospital admissions with diabetes and/or PAD from 2011 to 2016. The majority of the amputations were minor (61.7%; n = 3430), with a prevalence of 7.5 per 1000 and 40.4% (n = 2248) were major, with a prevalence of 4.9 per 1000. Geographic analysis found significant variation in risk for both major and minor amputation across the state, even after adjusting for the prevalence of risk factors. Analyses indicated an increased risk of amputation in the central and northeastern regions of West Virginia at the county level, although zip code-level patterns of amputation varied, with high-risk areas identified primarily in the northeastern and south central regions of the state. There is significant geographic variation in risk of amputation across West Virginia, even after adjusting for disease-related risk factors, suggesting priority areas for further investigation. The level of granularity obtained using advanced spatial analyses rather than traditional methods demonstrate the value of this approach, particularly when risk estimates are used to inform policy or public health intervention. [ABSTRACT FROM AUTHOR]

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro antimycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette–Guérin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains. [ABSTRACT FROM AUTHOR]

Summary: An unusual case of flail chest is presented. A 55year-old man with no history of trauma presented with back pain and breathlessness. He was found to have a mobile anterior chest wall, gross osteopaenia, a raised ESR and anaemia, but normal Bence–Jones protein. Bone marrow examination and serum electrophoresis confirmed a diagnosis of multiple myeloma. The flail chest was managed with analgesia and rest and the man was discharged home walking unassisted. [Copyright &y& Elsevier]

Objective: To evaluate the single dose pharmacokinetics of an intravenous dose of lorazepam in pediatric patients treated for status epilepticus (SE) or with a history of SE. Study design: Ten hospitals in the Pediatric Emergency Care Applied Research Network enlisted patients 3 months to 17 years with convulsive SE (status cohort) or for a traditional pharmacokinetics study (elective cohort). Sparse sampling was used for the status cohort, and intensive sampling was used for the elective cohort. Non-compartmental analyses were performed on the elective cohort, and served to nest compartmental population pharmacokinetics analysis for both cohorts. Results: A total of 48 patients in the status cohort and 15 patients in the elective cohort were enrolled. Median age was 7 years, 2 months. The population pharmacokinetics parameters were: clearance, 1.2 mL/min/kg; half-life, 16.8 hours; and volume of distribution, 1.5 L/kg. On the basis of the pharmacokinetics model, a 0.1 mg/kg dose is expected to achieve concentrations of approximately 100 ng/mL and maintain concentrations >30 to 50 ng/mL for 6 to 12 hours. A second dose of 0.05 mg/kg would achieve desired therapeutic serum levels for approximately 12 hours without excessive sedation. Age-dependent dosing is not necessary beyond using a maximum initial dose of 4 mg. Conclusions: Lorazepam pharmacokinetics in convulsive SE is similar to earlier pharmacokinetics measured in pediatric patients with cancer, except for longer half-life, and similar to adult pharmacokinetics parameters except for increased clearance. [ABSTRACT FROM AUTHOR]