Tuberculosis in prisons in sub-Saharan Africa--a potential time bomb.

No Abstrac

Application of highly portable MinION nanopore sequencing technology for the monitoring of nosocomial tuberculosis infection

Referral hospitals in sub-Saharan Africa concentrate large numbers of tuberculosis (TB) and multidrug-resistant TB (MDR-TB) patients, failed by community TB services. We have previously shown, from enhanced screening and through autopsy studies, a significant burden of missed TB infections at the University Teaching Hospital, Lusaka, Zambia, with many patients dying or being discharged without treatment. With minimal TB isolation facilities and minimal political will to invest in broader screening and isolation, the risk of nosocomial transmission is likely to be extremely high. Studies from other hospitals in low burden settings and in South Africa have shown that next generation sequencing (NGS) is a very powerful tool for rapidly sequencing whole TB genomes and comparing them to confirm or rule out nosocomial transmission. The established platforms for NGS analysis, such as Illumina, are very expensive, immobile, and require regular maintenance, making them a costly inclusion on a research proposal or programmatic intervention grant in Africa. MinION nanopore sequencing has changed the NGS landscape with cheap portable sequencers, rapid simple library preparation (15min), and automated real-time analysis tools. The application of highly portable MinION nanopore sequencing technology for the monitoring of nosocomial TB infection will be discussed. Preliminary data from our pediatric pneumonia study will demonstrate the detection of TB in induced sputum from children admitted to the University Teaching Hospital

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR.

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24h incubation in half-Fraser broth, 4h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1-5CFU/25g food sample and can be performed in 2 working days compared to up to 7days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n=175) and controls (n=31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food

tmRNA - a novel high-copy-number RNA diagnostic target - its application for Staphylococcus aureus detection using real-time NASBA

A real-time nucleic acid sequence-based amplification assay, targeting tmRNA, was designed for the rapid identification of Staphylococcus aureus. The selectivity of the assay was confirmed against a panel of 76 Staphylococcus strains and species and 22 other bacterial species. A detection limit of 1 cell equivalent was determined for the assay. A chimeric in vitro transcribed internal amplification control was developed and included in the assay. Application of the assay in natural and artificially contaminated unpasteurized (raw) milk enabled detection of 1-10 CFUS. aureus mL(-1) in 3-4 h, without the need for culture enrichment. Staphylococcus aureus was detected in all artificially contaminated milk samples (n=20) and none of the natural milk samples (n=20). Microbiological analysis of the natural milk samples was performed in parallel according to ISO 6888-3 and confirmed the absence of S. aureus. The method developed in this study has the potential to enable the specific detection of S. aureus in raw milk in a significantly shorter time frame than current standard methods. The assay further demonstrates the usefulness of tmRNA/ssrA as a nucleic acid diagnostic target

High Sensitivity DNA Detection Using Gold Nanoparticle Functionalised Polyaniline Nanofibres

Polyaniline (PANI) nanofibres (PANI-NF) have been modified with chemically grown gold nanoparticles to give a nanocomposite material (PANI-NF–AuNP) and deposited on gold electrodes. Single stranded capture DNA was then bound to the gold nanoparticles and the underlying gold electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen, Staphylococcus aureus (S. aureus), that causes mastitis. Significantly, cyclic voltammetry demonstrates that deposition of the gold nanoparticles increases the area available for DNA immobilisation by a factor of approximately 4. EPR reveals that the addition of the Au nanoparticles efficiently decreases the interactions between adjacent PANI chains and/or motional broadening. Finally, a second horseradish peroxidase (HRP) labelled DNA strand hybridises with the target allowing the concentration of the target DNA to be detected by monitoring the reduction of a hydroquinone mediator in solution. The sensors have a wide dynamic range, excellent ability to discriminate DNA mismatches and a high sensitivity. Semi-log plots of the pathogen DNA concentration vs. faradaic current were linear from 150 × 10−12 to 1 × 10−6 mol L−1 and pM concentrations could be detected without the need for molecular, e.g., PCR or NASBA, amplification