Detection and Imaging of Exposure-Related Metabolites and Xenobiotics in Hard Tissues by Laser Sampling and Mass Spectrometry.

The utility of two novel laser-based methods, laser ablation electrospray ionization (LAESI) and laser desorption ionization (LDI) from silicon nanopost array (NAPA), is explored via local analysis and mass spectrometry imaging (MSI) of hard tissues (tooth and hair) for the detection and mapping of organic components. Complex mass spectra are recorded in local analysis mode from tooth dentin and scalp hair samples. Nicotine and its metabolites (cotinine, hydroxycotinine, norcotinine, and nicotine) are detected by LAESI-MS in the teeth of rats exposed to tobacco smoke. The intensities of the detected metabolite peaks are proportional to the degree of exposure. Incorporating ion mobility separation in the LAESI-MS analysis of scalp hair enables the detection of cotinine in smoker hair along with other common molecular species, including endogenous steroid hormones and some lipids. Single hair strands are imaged by MALDI-MSI and NAPA-LDI-MSI to explore longitudinal variations in the level of small molecules. Comparing spectra integrated from NAPA-LDI-MSI and MALDI-MSI images reveals that the two techniques provide complementary information. There were 105 and 82 sample-related peaks for MALDI and NAPA, respectively, with an overlap of only 16 peaks, indicating a high degree of complementarity. Enhanced molecular coverage and spatial resolution offered by LAESI-MS and NAPA-LDI-MSI can reveal the distributions of known and potential biomarkers in hard tissues, facilitating exposome research.

Robust and sensitive amplicon-based whole-genome sequencing assay of respiratory syncytial virus subtype A and B.

Prevention of respiratory syncytial virus (RSV) infection is now a global health priority, with a long-acting monoclonal antibody and two RSV vaccines recently licenced for clinical use. Most licenced and candidate interventions target the RSV fusion (RSV-F) protein. New interventions may be associated with the spread of mutations, reducing susceptibility to antibody neutralization in RSV-F. There is a need for ongoing longitudinal global surveillance of circulating RSV strains. To achieve this large-scale genomic surveillance, a reliable, high-throughput RSV sequencing assay is required. Here we report an improved high-throughput RSV whole-genome sequencing (WGS) assay performed directly on clinical samples without additional enrichment, using a 4-primer-pool, short-amplicon PCR-tiling approach that is suitable for short-read sequencing platforms. Using upper respiratory tract (URT) RSV-positive clinical samples obtained from a sentinel network of primary care providers and from hospital patients (29.7% and 70.2%, respectively; n = 1,037), collected over the period 2019 to 2023, this assay had a threshold of approximately 4 × 103 to 8 × 103 copies/mL (RSV-B and RSV-A sub-types, respectively) as the lowest amount of virus needed in the sample to achieve >96% of whole-genome coverage at a high-quality level. Using a Ct value of 31 as an empirical cut-off, the overall assay success rate of obtaining >90% genome coverage at a read depth minimum of 20 was 96.83% for clinical specimens successfully sequenced from a total of 1,071. The RSV WGS approach described in this study has increased sensitivity compared to previous approaches and can be applied to clinical specimens without the requirement for enrichment. The updated approach produces sequences of high quality consistently and cost-effectively, suitable for implementation to underpin national programs for the surveillance of RSV genomic variation. IMPORTANCE: In this paper, we report an improved high-throughput respiratory syncytial virus (RSV) whole-genome sequencing (WGS) assay performed directly on clinical samples, using a 4-primer-pool, short-amplicon PCR-tiling approach that is suitable for short-read sequencing platforms. The RSV WGS approach described in this study has increased sensitivity compared to previous approaches and can be applied to clinical specimens without the requirement for enrichment. The updated approach produces sequences of high quality consistently and cost-effectively, suitable for implementation to underpin national and global programs for the surveillance of RSV genomic variation. The quality of sequence produced is essential for preparedness for new interventions in monitoring antigenic escape, where a single point mutation might lead to a reduction in antibody binding effectiveness and neutralizing activity, or indeed in the monitoring of retaining susceptibility to neutralization by existing and new interventions.