- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Poster Presentation
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- The Microbial Pangenome
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Protein families are the punchline of the pangenomes
More LessShort-read draft whole genome assemblies can contain many contigs and be impacted on by repeat regions, such as those caused by mobile element activity or inherently repetitive gene structure. Annotating such assemblies for gene content and functional activity can be challenging. This can be especially true if the predicted genes are fragmented across contigs, very large, repetitive or of unusual nucleotide content. Very high and low %GC genomes also come with additional issues. The Pfam domain database 1 is a widely-used large collection of protein families, each represented by multiple sequence alignments and Hidden Markov Models (HMMs). Rather than studying the predicted whole gene content of draft genomes, or presence/absence of specific genes in pan-core genome analyses, we examined predicted protein content by Pfam domain complement. Here we present Punchline, a workflow written in Python 3, to study the genetic content of pangenome assemblies including draft assemblies by looking at the complement of short protein domains. The domains can be used in statistical comparisons of Bacterial groups of interest as provided to the workflow of Punchline. In addition, we show the application of Punchline to specific genomic data.
1. The Pfam protein families database in 2019: S. El-Gebali, J. Mistry, A. Bateman, S.R. Eddy, A. Luciani, S.C. Potter, M. Qureshi, L.J. Richardson, G.A. Salazar, A. Smart, E.L.L. Sonnhammer, L. Hirsh, L. Paladin, D. Piovesan, S.C.E. Tosatto, R.D. Finn Nucleic Acids Research (2019) doi: 10.1093/nar/gky995
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Investigating the pangenomes of microbial eukaryotes
More LessPangenomes evolve in prokaryotes as a result of promiscuous horizontal transfer of genetic material and other rapid evolutionary processes. Although eukaryotes are generally under more restrictive evolutionary constraints than prokaryotes (e.g. lower levels of HGT), pangenomic structure has also been identified in plant, algal and fungal species. A number of methodologies for pangenome analysis have been published in recent years, but these methodologies are generally specifically-intended or optimized for prokaryotes. We have developed a software pipeline based around the previously-published PanOCT method of pangenome construction with additional pre- and post-processing methodologies tailored for eukaryote pangenome analysis. We have previously used this pipeline to analyze the pangenomes of model fungal species such as Saccharomyces cerevisiae, and we are currently testing our pipeline by constructing and analyzing the pangenome of the industrially-relevant yeast species Yarrowia lipolytica. Analysis of fungal pangenomes shows that core and accessory eukaryotic species genomes encompass a variety of phenotypes and suggest that gene duplication events play a larger role in eukaryote pangenome evolution than HGT.
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Oxford Nanopore sequencing elucidates a novel stx2f carrying prophage in a Shiga toxin producing Escherichia coli(STEC) O63:H6 associated with a case of haemolytic uremic syndrome (HUS)
More LessAimThe aim of this study was to use Oxford Nanopore sequencing to characterise a Shiga-toxin producing Escherichia coli(STEC) O63:H6 responsible for a recent case of haemolytic uremic syndrome (HUS).
MethodsSTEC isolate 377323 and a comparator stxnegative E. coli isolate of the same lineage and serotype (382634) were sequenced on an Oxford Nanopore minION R9.4 flow cell and an Illumina HiSeq 2500. Nanopore FAST5 data were basecalled and demultiplexed by Albacore. Assemblies were generated using Unicycler and polished using Nanopolish, Pilon and Racon before screening for prophages using PHASTER.
ResultsThe Shiga toxin subtype was confirmed to be stx2f, which is rarely associated with the onset of HUS in STEC. Comparing the two genomes, the stxnegative isolate harboured five prophages compared to eleven prophages in the STEC isolate, with only one in common. Both samples contained an intact locus of enterocyte effacement pathogenicity island. The Shiga toxin encoding prophage was 42.5 kb in size and predicted to contain 71 coding sequences. 377323 also encoded an 85kbp IncFIB plasmid, not present in 382634. This plasmid encodes a traconjugation cassette and the virulence bfpcassette encoding for a type IV bundle forming pili.
ConclusionLong read sequencing enabled the characterisation of a rare STEC and related E. coli IN relation to both prophage content and plasmid content and revealed significant differences in the pan-genome in addition to the acquisition of the novel Stx2f phage. This data contributes to the understanding of non-O157 STEC associated with HUS.
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Comparative pangenomics of Campylobacter species
More LessBacterial genome sequencing has become very popular over the last 15 years in the field of bacterial genomics and evolution with over >1000 genome sequences generated for the zoonotic Campylobacter jejuni and Campylobacter coli species. The rapid improvement of culture and sequencing techniques has led to isolation and sequencing of new Campylobacter species from a variety of sources including birds, mammals, reptiles and the environment. Inter-species recombination has been demonstrated for C. jejuni and C. coli, but little is known about recombination between other species in the genus. We analysed the population genomic structure of whole genome sequenced isolates from different Campylobacter species using pangenome and phylogenetic analyses to investigate core and accessory gene variation and putative gene function. Characterizing the extend of genome segregation among multiple Campylobacter species isolated from the same and different hosts improved understanding of how ecology (physical isolation) maintains species and the extent to which intrinsic mechanistic and adaptive barriers are eroded when species cohabit. This broadly defines the limit of interspecies recombination and the non-reducible pangenomically-defined species. This raises important questions about the nature of species specific recombinational barriers, the genes that constitute the inter-species mobilome and the emergence of zoonoses through reticulate evolution in agricultural animals.
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Lineage-specific evolution in Listeria monocytogenes detected by analysis of a panel of Swiss isolates from food and human origin
Listeria monocytogenes is the causative agent of Listeriosis, a foodborne infection and characterised by outbreaks with high mortality. In the past years, outbreak analysis has shifted from PFGE to core genome MLST (cgMLST) and a difference of ≤10 cgMLST alleles was identified as the most appropriate cut off. The cgMLST scheme contributes significantly to the analysis of outbreaks but is less suitable to provide insight in the overall population structure. In the present work, we characterize the differences in the evolution of lineage I and II and the impact they have on the identification of outbreak isolates starting from the genome sequences of a panel of 166 Swiss L. monocytogenes clinical and food isolates. We included in the analysis recently published genomes from Germany (414 isolates) and Holland (128 isolates). Using data of pairwise cgMLST and SNP differences of these 708 isolates, we can clearly identify the genetic diversity associated to outbreaks (≤10 differences), sublineages (≤150 differences) and lineages. The sublineages identified by SNP and cgMLST match the clustering of the PopPUNK software. When broken down for lineages, data show clearly that lineage II has a lower mutation rate within sublineages but a higher diversity between sublineages. Admixture analysis confirms that the increased diversity between sublineages in lineage II is due to horizontal gene transfer and recombination. While our data show that genome evolution is lineage-specific in L. monocytogenes, the cut off for the identification of outbreaks remains identical between both lineages.
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Pangenomic analysis of Staphylococcus pseudintermedius to better understand antimicrobial resistance profiles
More LessAlthough Staphylococcus pseudintermedius are commensal bacteria of dogs, they are also opportunistic pathogens, being the primary cause of canine skin and ear infections. Recently, more attention has been given to this species, due to the emergence of antimicrobial resistance. Further to this a methicillin resistant S. pseuintermedius was isolated from a human and was subsequently found to be multidrug resistant. To better understand the frequency of carriage of antimicrobial resistance, as well as the presence of multidrug resistance, we examined the pangenome of over 200 S. pseudintermedius isolates from across the globe. Focussing on methicillin resistance we were able to identify staphylococcal cassette chromosome mec (SCCmec) types unique to specific Bayesian Analysis of Population Structure (BAPS) groups, including groups which carried mec resistance genes independent of a known SCCmec element. In addition, we identified an SCCmec element, within isolates from North America, that shares 99 % nucleotide identity with a recently described non-typeable SCCmec element carried by Staphylococcus aureus isolated from a human in the Netherlands. Beyond methicillin resistance we found over 50 % of those strains analysed were putatively multidrug resistant (resistant to 3 or more antimicrobial drug classes). This highlights the diverse resistance determinants present in animal staphylococci and the importance of monitoring S. pseudintermedius.
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Evolution and spread of bacterial transposons
More LessTransposons are mobile genetic elements (MGE) that can carry additional genetic cargo nonessential for their own transposition. This cargo can include antibiotic or heavy metal resistance genes or those increasing metabolic plasticity. In addition to transposing across or between the chromosome and other replicons in a single cell, they can transfer between bacteria as passengers on conjugative plasmids that are capable of intercellular transfer. Transposons can be grouped on mechanisms of movement and by lengths of the bounding inverted repeats or of target site duplication created by transposition. Class II transposons, such as Tn3 and Tn501 utilise two-step replicative transposition involving a transposase, tnpA, and a resolvase, tnpR, gene. The Tn402/Tn5053 family has four genes, tniABQR, required for transposition, while Tn7 and relatives contain five (tnsABCDE). Despite their role in antimicrobial resistance dissemination and a detailed mechanistic view of transposition there have been no studies aimed at revealing the evolutionary history of transposon families by interrogating global genome sequence datasets. This is largely because the ability to search all existent bacterial sequences is non-trivial and only recently realised. We took a selection of 37 representative variants of the above transposon families and queried their nature and distribution across sequence space using bigsi (http://www.bigsi.io/), a searchable index of the bacterial ENA (455 632 datasets, Dec 2016) and Shovill (https://github.com/tseemann/shovill). Constrained by the sequence data that exists and the biases this may engender, this analysis provides broad insights into the prevalence and spread of important MGE.
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Resolving complex mobile genetic elements with nanopore sequencing
More LessSequence-based surveillance of antimicrobial resistance (AMR) is becoming increasingly prevalent – with the emergence of low-capital investment long-read technology in the form of Oxford Nanopore Technologies (ONT) MinION enabling such surveillance in infrastructure poor low-middle income countries. In such settings, the surveillance of mobile genetic elements, which can facilitate the rapid spread of beneficial AMR genotypes horizontally throughout bacterial populations, and across species boundaries may provide a relatively low-cost target for surveillance. In this study we used an ONT MinION to whole genome sequence E. coli isolates from Nairobi, Kenya for which Illumina data had previously been generated alongside producing whole genome sequences for novel isolates collected for a model surveillance project in Busia, Kenya. The Illumina sequenced isolates had been found to carry a consistently co-occurring set of antibiotic resistance genes (tetA, strAB, sul1, bla-TEM, and dfrA7), conferring resistance to five antibiotic classes. The exact genomic context of which could not be determined with Illumina sequence data alone. Whilst those isolates collected in Busia demonstrated phenotypic resistance to those classes. Initially suspected to be borne on regionally disseminated plasmids; the long-reads generated by the MinION allowed the full resolution of these genes across several closely associated transposable elements, which were further found to be integrated chromosomally, across these geographically independent isolates. This study demonstrates that value of long-read sequencing in the resolution of regionally important mobile genetic elements, simultaneously allowing further value to be added to previously produced long-read data.
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- Vaccines Against Bacterial Pathogens
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The identification of novel vaccine antigens and their potential role in preventing VTEC infections in children
More LessVTEC are a group of strains of E. coli, which cause severe bloody diarrhoea. VTEC infections can lead to the development of haemolytic uremic syndrome (HUS), which is the main cause of kidney failure among children. HUS can cause other life-long complications including seizures, bowel perforation and blindness. Ireland currently has the highest incidence of VTEC infections compared to any other European country. There are no vaccines available to protect children and immunocompromised adults against VTEC infections. Due to the risk of patients experiencing severe symptoms and complications, there is an urgent need for a vaccine against this infection. Bacterial proteins involved in host cell attachment have previously been shown to be efficacious prophylactic vaccine antigens for other infections. We have shown that an O157 strain, NCTC12900 has 1.3-fold higher binding to HT29 cells than the commensal strain, HS (P=0.0162). We have used a proteomic approach to identify the bacterial proteins involved in attachment to two human gastrointestinal epithelial cell lines, HT29 and Caco-2. We have identified seven host cell attachment proteins in VTEC, strain NCTC12900, that are not found in commensal strain, HS. These antigens will be examined for their ability to protect mice from VTEC challenge.
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Potential glycoengineered anti-Burkholderia vaccines by exploiting the bacterial O-glycosylation machinery
More LessPreviously, we discovered a protein O-glycosylation (ogc) cluster conserved in all Burkholderia species, which glycosylates proteins with a trisaccharide glycan. Sera from Burkholderia-infected patients produce anti-glycan antibodies, suggesting that the Burkholderia protein glycosylation pathway can be exploited for potential vaccine development. Here, we successfully produced two prototypes of anti-Burkholderia vaccines: a recombinant glycoprotein-based vaccine and an E. coli LPS-display vaccine. To generate the former, we constructed a plasmid carrying a chimeric gene encoding three glycosylation sequons fused to the cholera toxin B subunit. The presence of the glycan was observed in recombinant proteins expressed in B. cenocepacia parental strain, but not in proteins expressed by the glycosyltransferase-deficient ΔpglLstrain, as determined by SDS-PAGE and fluorescent lectin blots. For the development of an E. coli LPS-display vaccine, we constructed a plasmid expressing the ogc cluster, which was introduced into an E. coli strain unable to synthesize O-antigen but carrying the O-antigen ligase WaaL. Our results show that the LPS of this strain contained an additional moiety consistent with the B. cenocepacia trisaccharide glycan, as demonstrated by silver-stained LPS gels and lectin blot. This extra moiety was not detected in aΔwaaL mutant. These results suggest that the plasmid was able to provide the necessary functions for the synthesis and membrane translocation of the lipid-linked trisaccharide, which became a substrate for the WaaL ligase and incorporation into the E. coli LPS. Therefore, we demonstrate that the O-glycosylation pathway can be manipulated for the construction of potential anti-Burkholderia vaccines.
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- Virology Workshop: Antivirals and Vaccines
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Enhancing protective efficacy of poultry vaccines through targeted delivery of antigens to antigen presenting cells
More LessIn recent years, poultry production has been under constant threat of infectious diseases such as avian influenza and Newcastle disease resulting in the poultry trade reduction and zoonotic infection risk. Vaccination has become one of the important measures in controlling such diseases. One way of increasing the efficacy of these vaccines is to deliver specific antigens selectively targeting antigen presenting cells (APCs). This could be achieved by coupling APC receptor-specific antibody to the antigen of choice. To achieve this, we are targeting avian influenza hemagglutinin (HA) protein to the chicken dendritic cell (DC) surface receptors. We have recombinantly produced H9HA antigen fused to single chain fragment variable antibodies (scFv) against the chicken DC surface receptors. To assess the HA activity of our recombinantly produced H9HA fused to scFv antibody, hemagglutination assay was adopted. Our constructs were able to agglutinate chicken red blood cells and therefore retained HA activity. Furthermore, the scFv antibodies fused to HA were able to detect their respective antigens via western blot indicating that these scFv antibodies are functional. To further characterise our H9HA fused scFv antibodies in vitro, their binding ability to chicken DCs will be assessed using flow cytometry. Therefore, we have created functional H9HA antigen targeting to chicken DCs which we aim to use in developing enhanced vaccines towards avian influenza virus. In future, we will clone our constructs into herpesvirus of turkey to generate a recombinant viral vector vaccine which we hypothesise would enhance the immune response in chickens.
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Analysis of hepatitis C virus genotype 3 resistance to direct acting antivirals
Treatment of hepatitis C virus (HCV) with direct acting antivirals (DAAs), results in a sustained virological response (SVR) that varies according to the viral genotype. Genotype 3 is the second most prevalent in Brazil and presented the lowest response among all genotypes, this was associated with the occurrence of resistance substitutions in the viral genome. The aim of this study was to analyze genotype 3 viruses circulating in Brazilian patients who do not respond to treatment with Daclatasvir to investigate the presence of novel substitutions within NS5A, and to investigate the role of these substitutions in resistance to different NS5A inhibitors in vitro. A total of 60 patients have been analyzed, including 4 relapsers. Samples collected before (all patients) and after treatment (relapsing patients) were analysed by RNA extraction, cDNA synthesis, amplification by Nested-PCR and direct Sanger sequencing. Many previously undefined substitutions were indeed observed. These included S98G which, although detected in 15 % of pre-treatment samples, substitution appeared in 75 % of post-treatment samples from the non-responding patients and only in 10.7 % of responding individuals. For post-treatment samples in relapsing patients, this substitution had a prevalence of 50 %. Due to the high prevalence, this substitution showed potential to be associated with therapy failure. The phenotypes of this substitution and others identified in the study are being evaluated in the context of a genotype 3a subgenomic replicon and infectious virus to determine their effect on DAA resistance and/or potential fitness cost.
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Production and characterization of antibodies neutralizing H9N2 avian influenza virus
More LessMonoclonal antibodies (mAbs) that neutralize influenza A virus have been shown to be potent therapeutic reagents if used pre- or post – exposure to the pathogen. Whilst the majority of this research has focused on human antibody therapeutics, there is an urgent need for generation of antibodies able to neutralize avian influenza viruses (AIV). Several mAbs against H9N2 virus have previously been generated using mouse hybridomas. To facilitate development of passive immunization strategies, the variable chains of these antibodies were characterized in the context of isotype and species specific Fc fragments. Replacement of the IgG2 Fc region with an IgG1 Fc region enhanced neutralizing activity for one of the tested antibodies. Additionally, chicken chimeric antibodies were generated which showed comparable neutralization titres to those generated by the original hybridomas. To identify if antibodies could function as a single chain variable fragment antibodies (scFvs) these were produced in insect S2 cells. This confirmed that H9N2 virus can be neutralized by scFvs. However, an example with high HI titres but lost detectable neutralizing activity was found, possibly due to the loss of bivalent interaction between antigen and antibody. Antibodies showing superior neutralization activity in vitro will be subsequently tested for their potency in in vivo infection. We propose that passive immunization can reduce the impact of AIV in poultry by inducing immediate protection and bypassing immunocompromised individuals.
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Characterising the cell-mediated immune response to Lumpy skin disease virus
More LessLumpy skin disease (LSD), a high-impact disease of cattle and water buffalo, is a direct threat to the European cattle industry. The causative agent is the lumpy skin disease virus (LSDV), an enveloped dsDNA virus which belongs to the family Poxviridae. Affected cattle present multiple cutaneous nodules that are characteristic of a LSDV infection. The case fatality rate of LSD is low, however affected animals suffer substantial production losses including weight loss and reduced milk production. On a wider scale, LSD is a high consequence transboundary disease with mandated export restrictions imposed on affected countries leading to substantial economic costs. Currently, there is no cure for LSDV infected cattle. Hence, mass vaccination is an important component in minimizing the spread of LSDV. There are only a handful of commercially available live attenuated vaccines in the current market. These vaccines vary in efficacy, quality, and safety, which leaves some scope for further development. This improvement is hampered by a poor understanding of the immunology of LSDV, particularly the protective immune mechanisms. In this project, we are investigating the immune response to LSDV with a focus on characterising the cell-mediated immune response. Peripheral blood mononuclear cells from LSDV immunised/infected cattle and flow cytometry assays are used to detect the production of IFN-γ by CD4+T-helper cells and CD8+cytotoxic T cells in response to live LSDV stimulation. Our goal is to improve understanding of the immune response to LSDV in order to develop effective vaccines and control programs in the future.
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A synthetic biology approach for African horse sickness vaccine platforms
More LessAfrican horse sickness virus (AHSV) is the causative agent of African horse sickness (AHS), a highly fatal disease of equids. Currently, live attenuated vaccines are used to control AHS especially in South Africa, but they are in general subject to restrictions. We took advantage of previously published AHSV structural and antigenic data to simplify AHSV vaccine development using an AHSV-4 reverse genetics approach. We systematically substituted the tip and central domains of AHSV-4 outer capsid protein VP2 with the corresponding region from other serotypes to generate a chimeric protein. AHSV S2 chimeric and mono-reassortant viruses for all 9 serotypes were recovered by reverse genetic using AHSV-4 as backbone and used to immunise guinea pigs. Presence of neutralising antibody titres were determined using a fluorescence-based neutralisation assay. The exchange of the tip and central domain of VP2 switched the serotype specificity of the rescued chimeric viruses, however, sera from AHSV-4VP2DTip only neutralised the homologous AHSV-4 reference virus. Mono-reassortants, but not recombinant viruses containing chimeric VP2, induced antibodies with low levels of cross-neutralisation between phylogenetically related serotypes. Interestingly, most of the sera raised were able to neutralise AHSV-4, indicating the presence of other neutralising epitopes within the virus. These results raise the possibly of generating a single virus that affords protection against multiple serotypes. Our research highlights the ability to manipulate the AHSV genome to rapidly generate ‘synthetic’ viruses using a single platform approach for vaccine development.
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Application of Next Generation Phage Display technology to study cross reactivity in closely-related Flavivirus species
More LessFlaviviruses are a large family of viruses, some members of which cause human and veterinary disease and pose a potential risk of death. The transmission route is typically through the bite of arthropods such as ticks or mosquitoes and. They are a major cause of emerging and re-emerging viral infections. One of the major issues faced when carrying out serology diagnostics is the risk of cross reactive antibodies among closely related species. Next Generation Phage Display is a molecular biology technique combines phage display with next generation sequencing. A phage library is created by inserting peptide sequences into a phagemid vector. Each phage in the resultant library displays a particular peptide on its external surface and the resultant phagemid is packaged within the phage particle. The main advantage of phage display is linking the phenotype (peptide binding properties) with genotype (the peptide gene within the phagemid) Serum antibodies from flavivirus infected species are immobilised on a solid support and incubated with the phage library. Following washing steps to remove non-specific binding, the phage are rescued and propagated in bacteria. This process is called biopanning and is repeated up to 4 times. The phage genomes are sequenced using Ion Torrent sequencing and through analysing the sequences, the antigenic peptide regions can be identified. The most antigenically potent sites can then be used to create a diagnostic assay such an ELISA.
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- Virology Workshop: Clinical Virology
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An audit of the post-exposure management of rabies in Addenbrooke’s Hospital, Cambridge
More LessRabies is a clinically significant disease, caused by a neurotropic virus that is transmitted in the saliva of infected animals. The incubation period is exceptionally variable, followed by viral encephalitis, and almost always death. Overall rabies infection kills approximately 50 000 to 70 000 people/year. Control of the disease can be achieved by eliminating the animal reservoir or effective pre-exposure and post-exposure prophylaxis (PEP). Effective human rabies vaccines exist for pre-exposure immunization. These are normally recommended for people in high-risk occupations and travellers to rabies-affected areas. Although rabies is no longer endemic in the UK, it remains a significant problem in returning travellers. There are approximately 2000 PEP courses issued/year, 85–90 % for returning travellers and approximately 10–15 % for bat exposure in the UK. In this audit, we have evaluated the quality of the rabies PEP management in Addenbrooke’s Hospital, Cambridge over 3 years. The objectives of the audit were the correct risk assessment of the patient, timely prescription of PEP, as well as effective communication between clinical teams and record keeping. The standards were drawn from previously published literature. Although the standards relating to patient safety were met and exceeded, some drawbacks in communication and record keeping, as well as some non-auditable issues were identified. Additionally, a review of annual case numbers and peak periods of PEP referrals was used to identify key staff training periods. A shared experience with other issuing centres across the country could be used to optimise the approach to this highly clinically significant issue.
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A retrospective regional audit of Cytomegalovirus (CMV) laboratory diagnostics in Crohn’s/Colitis patients in Northern Ireland – ‘towards a diagnostic algorithm’
IntroductionConcurrent cytomegalovirus (CMV) in inflammatory bowel disease (IBD) related colitis, (Ulcerative Colitis (UC) or Crohn’s Disease (CD) is an important yet complex scenario associated with high rates of colectomy and other morbidity. This regional audit aimed to identify baseline standards for a virological diagnostic-based approach for the identification of those at risk of CMV reactivation (flare) in the setting of IBD.
MethodsRetrospective, cross-sectional study over a three year time-period, January 2010–March 2013, involving all five Northern Ireland HSC Trusts. Sample cohort n=277 IBD patients of which n=106 further grouped as SRC ‘severe acute colitis and/or steroid refractory colitis’. Seven audit standards were assessed including specimen(s) submission for CMV diagnostic analysis, antiviral therapy, ascertainment of colectomy rate, and result communication protocol.
FindingsAudit primarily found a need to better define test requesting protocols (optimal sample required for optimal serology and molecular diagnostics), and a need to work towards timeliness in both test requesting and result reporting. This audit also identified the high risk patient group (SRC) when test positive for CMV (PCR and/or histopathology) three times more likely to undergo colectomy (OR 3.16, 95 % CI (0.74, 13.21; P=0.06).
ConclusionsDiagnostic-based patient identification is optimal to identify those most at risk of CMV reactivation in the IBD setting. The serological IgG profile is key in risk assessment of potential reactivation, together with quantitative CMV PCR in colonic tissue, with/without supportive histopathology, as the most sensitive and timely means of identify those high risk patients.
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Use of hyperattenuated poliovirus as a replacement for Sabin or wild-type strains for laboratory assays in a post eradication world
More LessPoliovirus serotype 2 (PV2) has been declared eradicated and was removed from the live vaccine programme in 2016. WHO have developed the Global Action Plan (GAPIII) to describe containment conditions for laboratory work with poliovirus after eradication, and in the UK, all PV2 has been reclassified to hazard group 3 (HG3). Laboratory work involving live poliovirus will continue to be required years after eradication for the purposes of: vaccine testing; environmental surveillance; immunoglobulin testing; and for the development of new public health interventions. Compliance with GAPIII and HG3 containment conditions would impose financial burden and failure would lead to disruption to essential activities. Development of safer poliovirus strains for use outside of containment could overcome these issues. The S19 poliovirus strain, designed to be hyperattenuated and extremely genetically stable, was used as a cassette for the introduction of capsid proteins of other strains and serotypes (Knowlson et al. 2015), originally for vaccine production. S19 viruses are unable to infect transgenic mice (by intraspinal inoculation) or non-human primates (by mouth) at very high doses and are unlikely to infect humans at biological temperature. PV2 S19 strains have recently been approved for use outside of GAPIII containment requirements by the Containment Advisory Group. Validation of strains for laboratory assays and a highly sensitive QC assay based on NGS will be described.
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Macrolide-resistant Mycoplasma genitalium in male and female patients attending a Northern Irish Genitourinary Medicine Clinic
More LessBackgroundMycoplasma genitalium (MG) can cause urethritis in men and pelvic inflammatory disease in women. Despite the increasing rate of MG resistance to first-line macrolide treatment due to 23S rRNA gene mutations, the availability of testing remains limited in UK laboratories. This study obtained preliminary data on the rate of macrolide resistant strains circulating in N. Ireland.
MethodsClinical specimens from 1052 patients attending a Genitourinary Medicine (GUM) clinic in Belfast from April 2017 to April 2018 were screened using an in-house MG PCR assay. MG positive samples were subsequently tested using Resistance Plus™ MG assay (Speedx, Australia) to detect 23S rRNA gene mutations associated with macrolide resistance.
ResultsAmongst all study samples, 3.9 % (n=41) tested positive for MG. Out of these 41.5 % (n=17) had a 23S rRNA mutation. The rate of MG was highest in rectal swabs (8.13 %) followed by vaginal swabs (4.96 %) and then male urines (2.85 %). The rate of 23 S rRNA mutations associated with macrolide resistance in positive vaginal swabs was 21.4 % whereas the rate amongst urine and rectal swabs was at least 50 %.
ConclusionData from this study adds to the evidence base to perform MG testing in risk groups in N. Ireland to improve patient outcomes and reduce antimicrobial resistance.
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