Browsing by Author "Hassan, Marwa M."
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- Detection of Rickettsia in ticks using loop-mediated isothermal amplification (LAMP)Publication . Lansdell, Samantha; Hassan, Marwa M.; La Ragione, Roberto; Betson, Martha; Nuncio, MS; Lopes de Carvalho, Isabel; Zé-Zé, Líbia; de Sousa, Rita; Cutler, Sally; Davis, Joshua S.Objectives: The objective of the study was to develop a Loop-mediated Isothermal Amplification (LAMP) assay for screening of Rickettsia species circulating in ticks using the citrate synthase gene (gltA). The LAMP assay employed portable visualisation methods, making the assay more field-suitable. Furthermore, prior methods have not used gltA as the target, despite proven success in Polymerase Chain Reaction (PCR) methods. Methods: Using an alignment of 72 DNA sequences (comprised of 21 Rickettsia species) from GenBank we designed a novel set of gltA LAMP primers. Evaluation used DNA from 12 Rickettsia species as positive controls (extracted from cultures or naturally infected ticks) alongside a panel of negative controls representing different bacterial species. Subsequently this assay was used to screen 295 Ixodes ricinus and 24 I. hexagonus ticks collected from the UK (including northern and southern England and northern Scotland). Results: LAMP successfully detected 11 out of 12 (91.7%) Rickettsia species, excluding Rickettsia akari. Among 319 ticks collected in the UK, three were positive for Rickettsia (0.9%). All three positives were I. ricinus ticks, while none of the 24 I. hexagonus ticks were positive. Results were confirmed using a published PCR method. Sanger sequencing of PCR amplicons generated for each positive tick showed that they were all R. helvetica. Conclusions: This study introduces a novel field-applicable LAMP protocol for efficient Rickettsia screening in ticks to better assess its prevalence and consequent health risks. Furthermore, this assay has proven suitability for rickettsial detection in I. ricinus ticks, which has been reported as unsuccessful in previous European studies.
- Mapping the evidence of the effects of environmental factors on the prevalence of antibiotic resistance in the non-built environment: Protocol for a systematic evidence mapPublication . Gardner, Brian; Betson, Martha; Cabal Rosel, Adriana; Caniça, Manuela; Chambers, Mark A.; Contadini, Francesca M.; Gonzalez Villeta, Laura C.; Hassan, Marwa M.; La Ragione, Roberto M.; de Menezes, Alexandre; Messina, Davide; Nichols, Gordon; Olivença, Daniel V.; Phalkey, Revati; Prada, Joaquin M.; Ruppitsch, Werner; Santorelli, Lorenzo A.; Selemetas, Nick; Tharmakulasingam, Mukunthan; M. van Vliet, Arnoud H.; Woegerbauer, Markus; Deza-Cruz, Iñaki; Lo Iacono, GiovanniBackground: Human, animal, and environmental health are increasingly threatened by the emergence and spread of antibiotic resistance. Inappropriate use of antibiotic treatments commonly contributes to this threat, but it is also becoming apparent that multiple, interconnected environmental factors can play a significant role. Thus, a One Health approach is required for a comprehensive understanding of the environmental dimensions of antibiotic resistance and inform science-based decisions and actions. The broad and multidisciplinary nature of the problem poses several open questions drawing upon a wide heterogeneous range of studies. Objective: This study seeks to collect and catalogue the evidence of the potential effects of environmental factors on the abundance or detection of antibiotic resistance determinants in the outdoor environment, i.e., antibiotic resistant bacteria and mobile genetic elements carrying antibiotic resistance genes, and the effect on those caused by local environmental conditions of either natural or anthropogenic origin. Methods: Here, we describe the protocol for a systematic evidence map to address this, which will be performed in adherence to best practice guidelines. We will search the literature from 1990 to present, using the following electronic databases: MEDLINE, Embase, and the Web of Science Core Collection as well as the grey literature. We shall include full-text, scientific articles published in English. Reviewers will work in pairs to screen title, abstract and keywords first and then full-text documents. Data extraction will adhere to a code book purposely designed. Risk of bias assessment will not be conducted as part of this SEM. We will combine tables, graphs, and other suitable visualisation techniques to compile a database i) of studies investigating the factors associated with the prevalence of antibiotic resistance in the environment and ii) map the distribution, network, cross-disciplinarity, impact and trends in the literature.
- Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal AmplificationPublication . Higgins, Owen; Chueiri, Alexandra; O'Connor, Louise; Lahiff, Sinéad; Burke, Liam; Morris, Dearbhaile; Pfeifer, Nicola Maria; Santamarina, Belén González; Berens, Christian; Menge, Christian; Caniça, Manuela; Manageiro, Vera; Kisand, Veljo; Hassan, Marwa M.; Gardner, Brian; van Vliet, Arnoud H.M.; La Ragione, Roberto M.; Gonzalez-Zorn, Bruno; Smith, Terry J.Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The blaCTX-M-1 and blaCTX-M-15 genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of blaCTX-M-1 and blaCTX-M-15. Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for blaCTX-M-1 (n = 18), blaCTX-M-15 (n = 35), and other closely related blaCTX-Ms (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli blaCTX-M. Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for blaCTX-M-1 and blaCTX-M-15, respectively, and E. coli blaCTX-M-1 was identified in all blaCTX-M positive porcine fecal samples tested.