Declining Artesunate-Mefloquine Efficacy against Falciparum Malaria on the Cambodia–Thailand Border

Emerging resistance in Southeast Asia raises concern over possible spread or similar evolution of resistance to other artemisinin-based combination therapies in Africa

Metal worker’s lung: spatial association with Mycobacterium avium

Background Outbreaks of hypersensitivity pneumonitis (HP) are not uncommon in workplaces where metal working fluid (MWF) is used to facilitate metal turning. Inhalation of microbe-contaminated MWF has been assumed to be the cause, but previous investigations have failed to establish a spatial relationship between a contaminated source and an outbreak. Objectives After an outbreak of five cases of HP in a UK factory, we carried out blinded, molecular-based microbiological investigation of MWF samples in order to identify potential links between specific microbial taxa and machines in the outbreak zone. Methods Custom-quantitative PCR assays, microscopy and phylogenetic analyses were performed on blinded MWF samples to quantify microbial burden and identify potential aetiological agents of HP in metal workers. Measurements and main results MWF from machines fed by a central sump, but not those with an isolated supply, was contaminated by mycobacteria. The factory sump and a single linked machine at the centre of the outbreak zone, known to be the workstation of the index cases, had very high levels of detectable organisms. Phylogenetic placement of mycobacterial taxonomic marker genes generated from these samples indicated that the contaminating organisms were closely related to Mycobacterium avium. Conclusions We describe, for the first time, a close spatial relationship between the abundance of a mycobacterium-like organism, most probably M. avium, and a localised outbreak of MWF-associated HP. The further development of sequence-based analytic techniques should assist in the prevention of this important occupational disease

Regulatory Hotspots in the Malaria Parasite Genome Dictate Transcriptional Variation

The determinants of transcriptional regulation in malaria parasites remain elusive. The presence of a well-characterized gene expression cascade shared by different Plasmodium falciparum strains could imply that transcriptional regulation and its natural variation do not contribute significantly to the evolution of parasite drug resistance. To clarify the role of transcriptional variation as a source of stain-specific diversity in the most deadly malaria species and to find genetic loci that dictate variations in gene expression, we examined genome-wide expression level polymorphisms (ELPs) in a genetic cross between phenotypically distinct parasite clones. Significant variation in gene expression is observed through direct co-hybridizations of RNA from different P. falciparum clones. Nearly 18% of genes were regulated by a significant expression quantitative trait locus. The genetic determinants of most of these ELPs resided in hotspots that are physically distant from their targets. The most prominent regulatory locus, influencing 269 transcripts, coincided with a Chromosome 5 amplification event carrying the drug resistance gene, pfmdr1, and 13 other genes. Drug selection pressure in the Dd2 parental clone lineage led not only to a copy number change in the pfmdr1 gene but also to an increased copy number of putative neighboring regulatory factors that, in turn, broadly influence the transcriptional network. Previously unrecognized transcriptional variation, controlled by polymorphic regulatory genes and possibly master regulators within large copy number variants, contributes to sweeping phenotypic evolution in drug-resistant malaria parasites

Molecular techniques and their limitations shape our view of the holobiont

It is now recognised that the biology of almost any organism cannot be fully understood without recognising the existence and potential functional importance of associated microbes. Arguably, the emergence of this holistic viewpoint may never have occurred without the development of a crucial molecular technique, 16S rDNA amplicon sequencing, which allowed microbial communities to be easily profiled across a broad range of contexts. A diverse array of molecular techniques are now used to profile microbial communities, infer their evolutionary histories, visualise them in host tissues, and measure their molecular activity. In this review, we examine each of these categories of measurement and inference with a focus on the questions they make tractable, and the degree to which their capabilities and limitations shape our view of the holobiont

Profiling the host response and microbiome in pulmonary non-tuberculous mycobacterial infection

Abstract Background: Pulmonary non-tuberculous mycobacterial (NTM) disease is a challenging and increasingly prevalent infection. The factors which govern the development of clinical NTM disease and its subsequent clinical course are poorly understood. Aims: The aims of this thesis have been i) to investigate the host response to NTM infection using whole blood gene expression profiling, ii) to characterise the lung microbiome associated with pulmonary NTM disease using sequencing of the bacterial 16S rRNA gene and iii) to develop a culture-free method of characterising the mycobacterial communities present in the lung. Methods, Results & Conclusions: Gene expression analysis found that in NTM disease there was reduced expression the gene encoding interferon-γ, a key cytokine in the response to mycobacterial infection, as well as a number of genes associated with T-cell receptor signalling. In NTM disease survival was associated with genes involved in T- and B-cell function and mortality with genes related to the innate immune response and inflammation. Characterisation of the microbiome in both NTM disease and controls revealed an abundance of Proteobacteria. Compared with controls, dominance by Pseudomonas was less common in NTM disease, whereas Stenotrophomonas and Achromobacter dominance was more common. Several genera including Stenotrophomonas, Achromobacter, Burkholderia and Staphylococcus were more abundant in NTM disease. Sequencing of the hsp65 gene was used to characterise mycobacterial communities in the lung. Mycobacteria were found in all samples regardless of their isolation in culture or the presence of clinical NTM disease. Multiple species of mycobacteria were present in almost all samples, in contrast to culture which did not detect more than one species in any sample. Communities were often dominated by one species, with a lower diversity seen in subjects with clinical disease. Within subjects with NTM disease, lower diversity was seen in those who died and those with low IFNG expression.Open Acces

Non-tuberculous mycobacterial pulmonary disease

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Profiling mycobacterial communities in pulmonary nontuberculous mycobacterial disease.

The diagnosis of pulmonary non-tuberculous mycobacterial disease (pNTM) is dependent on the isolation of NTM in culture, which is prone to overgrowth and contamination and may not capture the diversity of mycobacteria present, including rare or unidentified species. This study aimed to develop a culture independent method of detecting and identifying mycobacteria from sputum samples using partial sequencing of the hsp65 gene. DNA was extracted from sputum samples from subjects with pNTM and disease controls. Multiplexed partial sequencing of the hsp65 gene was performed using the Illumina MiSeq and custom primers. A reference database of hsp65 sequences was created for taxonomy assignment. Sequencing results were obtained from 42 subjects (31 cases, 11 controls). Mycobacterial sequences were identified in all subjects. In 90.5% of samples more than one species was found (median 5.5). The species isolated in culture was detected by sequencing in 81% of subjects and was the most abundant species in 62%. The sequencing of NTM from clinical samples reveals a far greater diversity than conventional culture and suggests NTM are present as communities rather than a single species. NTM were found to be present even in the absence of isolation in culture or clinical disease

Latent class analysis to define radiological subgroups in pulmonary nontuberculous mycobacterial disease

Abstract Background Nontuberculous mycobacterial (NTM) pulmonary disease has conventionally been classified on the basis of radiology into fibrocavitary and nodular-bronchiectatic disease. Whilst being of great clinical utility, this may not capture the full spectrum of radiological appearances present. The aim of this study was to use latent class analysis (LCA) as an unbiased method of grouping subjects with NTM-pulmonary disease based on their CT features and to compare the clinical characteristics of these groups. Methods Individuals with NTM-pulmonary disease were recruited and a contemporaneous CT scan obtained. This was scored using an NTM-specific scoring system. LCA was used to identify groups with common radiological characteristics. The analysis was then repeated in an independent cohort. Results Three classes were identified in the initial cohort of 85 subjects. Group 1 was characterised by severe bronchiectasis, cavitation and aspergillomas, Group 2 by relatively minor radiological changes, and Group 3 by predominantly bronchiectasis only. These findings were reproduced in an independent cohort of 62 subjects. Subjects in Group 1 had a lower BMI and serum albumin, higher serum CRP, and a higher mortality. Conclusions These findings suggest that NTM-pulmonary may be divided into three radiological subgroups, and that important clinical and survival differences exist between these groups