2025/01/01 by R Childs Hunt · 1 voice
Biochemistry, Genetics and Molecular Biology · Medicine · #Bacterial biofilms and quorum sensing #Legionella and Acanthamoeba research #Ocular Infections and Treatments
paper · doi:10.17037/pubs.04678296
openalex publication_date 2025/01/01 · openalex created_date 2026/01/29 · openalex updated_date 2026/07/01
Free living amoebae of the genus Acanthamoeba are the causative agent of Acanthamoeba Keratitis (AK), a sight threatening infection of the cornea. Acanthamoeba is a diverse genus for which several morphologically described species, and 23 distinct genotypes defined at the 18S rRNA gene locus, are recognised. There is some evidence that diversity amongst the causative Acanthamoeba spp. is responsible for the variable outcome of AK treatment. Through its role in providing a reference service for identification of Acanthamoeba spp., the Diagnostic Parasitology Laboratory (DPL) at the London School of Hygiene & Tropical Medicine maintains an archive of clinical sample material, extracted DNA and cultured isolates. This thesis presents an investigation into the diversity of phenotype and genotype among these samples using morphological, phylogenetic and whole-genome approaches. Clonal isolates were derived from nine original clinical isolates and morphologically characterised. Morphological classification was in many cases inconclusive and has been shown to be unreliable by other authors. As molecular approaches are more specific and higher throughput, the clonal isolates, and additional uncloned isolates were further characterised with molecular techniques, PCR-RFLP and 18S rRNA gene genotyping. The diagnostic “Acanthamoeba specific amplimer” (ASA.S1) fragment of the 18S rRNA gene was sequenced using Sanger sequencing, allowing for a genotype to be provided for 29 DPL isolates. In pursuit of more detailed and higher throughput genetic characterisation of both cultured isolates and directly from clinical samples, a PCR, sequencing and bioinformatics pipeline was developed. The pipeline sought to obtain longer 18S rRNA gene sequences using the Oxford Nanopore Technologies’ MinION sequencer and software packages, as well as open-source software such as NGSpeciesID to produce high-quality consensus sequences. The pipeline enabled 66 full-length 18S rRNA sequences to be obtained from DPL samples and showed promising capabilities to obtain accurate sequences from polyclonal samples and from DNA extracted directly from clinical isolates without in vitro propagation. Acanthamoeba genotypes T4A, T4B, T4C, T4E, T3 and T6 were found to be represented amongst the isolates using the full-length sequences. Additionally, some of these isolates were typed as T4D and T4F when using the shorter ASA.S1 fragment for the phylogenetic analysis in concordance with reports that the two fragment sizes can produces different phylogenies. A reference-genome was assembled for a clonal cultured isolate, GP39-C1, originally derived from a corneal scrape sample. GP39-C1 was found to have the rare genotype T6, for which no reference genome has been published. Four different assemblies were produced, the most complete of which was estimated to be 94.1% complete. It was found that the assemblies with the highest estimated completeness also had the highest number of duplicate genes. The output genomes were compared with those of Acanthamoeba castellanii C3 and Acanthamoeba palestinensis Reich. GP39-C1 was found to be divergent from these genomes by 17.49% and 10.80% respectively. These pipelines and their outputs represent an enhanced capability for the DPL to provide genotypes and whole genome sequences for clinically derived samples and isolates. These data should be linked to clinical outcome and drug susceptibility, to better characterise the role that genetic diversity amongst Acanthamoeba spp. plays in the variable treatment outcome that patients experience.