A tuberculosis molecular bacterial load assay (TB-MBLA)

Funding: European and Developing Countries Clinical Trials Partnership (EDCTP) – Pan African Biomarker Expansion program (PanBIOME) grant SP.2011.41304.008. Support was also obtained the University of St Andrews School of Medicine research grant.Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers’ safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 °C, 85 °C, or 95 °C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 µL of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of <1 log10eCFU/mL (i.e., <10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.Publisher PDFPeer reviewe

Haem-iron plays a key role in the regulation of the Ess/Type VII secretion system of <i>Staphylococcus aureus</i> RN6390

This study was supported by the Wellcome Trust (through Investigator Award 10183/Z/15/Z to T. P. and through Clinical PhD studentship support to C. P. H. through grant 104241/z/14/z), the Biotechnology and Biological Sciences Research Council and the Medical Research Council (through grants BB/H007571/1 and MR/M011224/1, respectively).The Staphylococcus aureus type VII protein secretion system (T7SS) plays important roles in virulence and intra-species competition. Here we show that the T7SS in strain RN6390 is activated by supplementing the growth medium with haemoglobin, and its cofactor haemin (haem B). Transcript analysis and secretion assays suggest that activation by haemin occurs at a transcriptional and a post-translational level. Loss of T7 secretion activity by deletion of essC results in upregulation of genes required for iron acquisition. Taken together these findings suggest that the T7SS plays a role in iron homeostasis in at least some S. aureus strains.Publisher PDFPeer reviewe

SFX-01 in hospitalised patients with community-acquired pneumonia during the COVID-19 pandemic : a double-blind, randomised, placebo-controlled trial

We acknowledge the members of the STAR-COVID data monitoring committee: Aran Singanayagam (Imperial College, London, UK), Timothy Hinks (University of Oxford, Oxford, UK), Oriol Sibila (Hospital Clinic, Barcelona, Spain), Alex McConnachie (University of Glasgow, Glasgow, UK) and Petra Rauchhaus (University of Dundee, Dundee, UK). This trial was delivered by Tayside Clinical Trials Unit, a UKCRC registered clinical trials unit. Thanks to Clare Clarke, Jennifer Taylor, Angela Strachan, Heather Loftus and Jodie Strachan (Ninewells Hospital and Medical School, Dundee, UK) and Diane Cassidy (University of Dundee). We thank all study participants and their families.Peer reviewe

Developing a novel molecular bacterial load assay to improve clinical management of Mycobacterium abscessus infections

Mycobacterium abscessus (M. abscessus) is one of the most common rapid growing non- tuberculous mycobacteria (NTM) causing pulmonary disease (PD). Treatment involves long and toxic multi-drug regimens with uncertain benefit. It is difficult to assess antibiotic efficacy as current treatment monitoring depends on semi-quantitative culture of serial clinical samples that takes weeks to provide results. This thesis describes the development of the first molecular, quantitative treatment monitoring tool for NTM-PD. The M. abscessus molecular bacterial load assay (MBLA) assay which we developed provides results within a working day. The M. abscessus MBLA targets the hypervariable portions of 16S and pre-16S rRNA using real-time quantitative PCR to solely quantify the viable organisms from patients’ sputum samples. Both RNA targets showed a strong correlation with cell viability, having >90% degradation within four weeks following cell death. The M. abscessus MBLA uses a standard curve to provide bacterial load quantification. An investigation into the impact of antibiotics used for M. abscessus- PD treatment on the standard curve revealed a correlation between the 16S rRNA quantification and bacterial load in the absence and presence of antibiotics. The potential of using pre-16S rRNA to 16S rRNA ratio as a measure of metabolic activity within a bacterial population was explored, showing that the ratio followed the growth trends of M. abscessus in the absence and presence of antibiotics. This thesis resulted in a treatment monitoring tool selective for M. abscessus-chelonae group species with an efficiency of 94% and limit of detection of log 10¹ CFU/ml. Preliminary clinical validation was limited by a small number of available samples from patients with M. abscessus- PD, but the assay accurately reported 6/8 positive results and 36/36 negative results against a gold standard of sputum culture. Larger clinical studies will be undertaken to fully evaluate the clinical utility of the test

A tuberculosis molecular bacterial load assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers’ safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 °C, 85 °C, or 95 °C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 µL of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &lt;1 log10eCFU/mL (i.e., &lt;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation

Haem-iron plays a key role in the regulation of the Ess/type VII secretion system of Staphylococcus aureus RN6390 (dataset)

RNA-seq data deposited atthe European Nucleotide Archive with accession number ERP00927

Haem-iron plays a key role in the regulation of the Ess/type VII secretion system of Staphylococcus aureus RN6390 (dataset)

RNA-seq data deposited atthe European Nucleotide Archive with accession number ERP00927

Haem-iron plays a key role in the regulation of the Ess/type VII secretion system of Staphylococcus aureus RN6390

RNA-seq data deposited at the European Nucleotide Archive with accession number ERP00927

Haem-iron plays a key role in the regulation of the Ess/type VII secretion system of Staphylococcus aureus RN6390

RNA-seq data deposited at the European Nucleotide Archive with accession number ERP00927