Biology subjects

Ling, C. L.

Publications and source records attributed to Ling, C. L..

4 recordsLinked to original sources

bioRxiv2022

Target-enrichment sequencing yields valuable genomic data for difficult-to-culture bacteria of public health importance

Genomic data contribute invaluable information to the epidemiological investigation of pathogens of public health importance. However, whole genome sequencing (WGS) of bacteria typically relies on culture, which represents a major hurdle for generating such data for a wide range of species for which culture is challenging. In this study, we assessed the use of culture-free target-enrichment sequencing as a method for generating genomic data for two bacterial species: 1) Bacillus anthracis, which causes anthrax in both people and animals and whose culture requires high level containment facilities; and 2) Mycoplasma amphoriforme, a fastidious emerging human respiratory pathogen. We obtained high quality genomic data for both species directly from clinical samples, with sufficient coverage (>15X) for confident variant calling over at least 80% of the baited genomes for over two thirds of the samples tested. Higher qPCR cycle threshold (Ct) values (indicative of lower pathogen concentrations in the samples), pooling libraries prior to capture, and lower captured library concentration were all statistically associated with lower capture efficiency. The Ct value had the highest predictive value, explaining 52% of the variation in capture efficiency. Samples with Ct values [≤] 30 were over 6 times more likely to achieve the threshold coverage than those with a Ct > 30. We conclude that target-enrichment sequencing provides a valuable alternative to standard WGS following bacterial culture and creates opportunities for an improved understanding of the epidemiology and evolution of many clinically important pathogens for which culture is challenging. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Scripts used in this study can be accessed on GitHub at https://github.com/tristanpwdennis/bactocap. All sequence data generated during this study have been deposited in the European Nucleotide Archive (ENA) Sequence Read Archive (SRA) under project accession numbers PRJEB46822 (B. anthracis) and PRJEB50216 (M. amphoriforme). Accession numbers for individual samples, along with metadata, laboratory parameters and sequence quality metrics, are available at the University of Glasgows data repository, Enlighten, at http://dx.doi.org/10.5525/gla.researchdata.1249.

molecular biology↗

bioRxiv2020

Reverse transcription quantitative PCR to detect low density malaria infections

Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method. Here we evaluated a real-time quantitative reverse transcription PCR (qRT-PCR) method that significantly reduces the required sample volume compared to uPCR. To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were compared using uPCR and qRT-PCR. Plasmodium spp. qRT-PCR confirmed 18 of 21 uPCR Plasmodium falciparum positives, while P. falciparum specific qRT-PCR confirmed 17 of the 21 uPCR P. falciparum positives. Combining both qRT-PCR results increased the sensitivity to 100% and specificity was 95.1%. These results show that malaria detection in areas of low transmission and LDMI, can benefit from the increased sensitivity of qRT-PCR especially where sample volume is limited.

microbiology↗

bioRxiv2019

Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from south and southeast Asia.

BackgroundK. pneumoniae is a leading cause of blood stream infection (BSI). Strains producing extended spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial resistant (AMR) K. pneumoniae, and also for the characteristically antimicrobial sensitive, community-acquired hypervirulent strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. MethodsWe conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate. FindingsK. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common amongst South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes amongst K. pneumoniae. InterpretationSouth and Southeast Asia are high-risk regions for the emergence of AMR and convergent AMR-hypervirulent K. pneumoniae. Enhanced surveillance efforts, reporting STs, AMR and virulence information are urgently required to monitor this public health threat. FundingThis work was supported by the Wellcome Trust (grant #206194 to Wellcome Sanger Institute) and the Bill and Melinda Gates Foundation, Seattle (grant OPP1175797 to KEH). KEH is supported by a Senior Medical Research Fellowship from the Viertel Foundation of Australia. DAB and PNN are supported by the Wellcome Trust.

microbiology↗

bioRxiv2018

Candidatus Ornithobacterium hominis sp. nov.: insights gained from draft genomes obtained from nasopharyngeal swabs

Candidatus Ornithobacterium hominis sp. nov. represents a new member of the Flavobacteriaceae detected in 16S rRNA gene surveys from Southeast Asia, Africa and Australia. It frequently colonises the infant nasopharynx at high proportional abundance, and we demonstrate its presence in 42% of nasopharyngeal swabs from 12 month old children in the Maela refugee camp in Thailand. The species, a Gram negative bacillus, has not yet been cultured but the cells can be identified in mixed samples by fluorescent hybridisation. Here we report seven genomes assembled from metagenomic data, two to improved draft standard. The genomes are approximately 1.9Mb, sharing 62% average amino acid identity with the only other member of the genus, the bird pathogen Ornithobacterium rhinotracheale. The draft genomes encode multiple antibiotic resistance genes, competition factors, Flavobacterium johnsoniae-like gliding motility genes and a homolog of the Pasteurella multocida mitogenic toxin. Intra- and inter-host genome comparison suggests that colonisation with this bacterium is both persistent and strain exclusive.

genomics↗