Biochemistry and Pharmacology - Research Publications

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    Global expansion of Mycobacterium tuberculosis lineage 4 shaped by colonial migration and local adaptation
    Brynildsrud, OB ; Pepperell, CS ; Suffys, P ; Grandjean, L ; Monteserin, J ; Debech, N ; Bohlin, J ; Alfsnes, K ; Pettersson, JO-H ; Kirkeleite, I ; Fandinho, F ; da Silva, MA ; Perdigao, J ; Portugal, I ; Viveiros, M ; Clark, T ; Caws, M ; Dunstan, S ; Phan, VKT ; Lopez, B ; Ritacco, V ; Kitchen, A ; Brown, TS ; van Soolingen, D ; O'Neill, MB ; Holt, KE ; Feil, EJ ; Mathema, B ; Balloux, F ; Eldholm, V (AMER ASSOC ADVANCEMENT SCIENCE, 2018-10)
    On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.
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    The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens
    Tran, VTN ; Karkey, A ; Dongol, S ; Hang, NT ; Dunstan, S ; Holt, K ; Le, TPT ; Campbell, JI ; Tran, TC ; Nguyen, VVC ; Arjyal, A ; Koirala, S ; Basnyat, B ; Dolecek, C ; Farrar, J ; Baker, S (BMC, 2010-05-21)
    BACKGROUND: PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive. METHODS: We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients. RESULTS: The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75) and sensitivity of 53.9% (69/128) on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles. CONCLUSIONS: Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.
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    The STRATAA study protocol: a programme to assess the burden of enteric fever in Bangladesh, Malawi and Nepal using prospective population census, passive surveillance, serological studies and healthcare utilisation surveys
    Darton, TC ; Meiring, JE ; Tonks, S ; Khan, MA ; Khanam, F ; Shakya, M ; Thindwa, D ; Baker, S ; Basnyat, B ; Clemens, JD ; Dougan, G ; Dolecek, C ; Dunstan, SJ ; Gordon, MA ; Heyderman, RS ; Holt, KE ; Pitzer, VE ; Qadri, F ; Zaman, K ; Pollard, AJ (BMJ PUBLISHING GROUP, 2017-06)
    INTRODUCTION: Invasive infections caused by Salmonella enterica serovar Typhi and Paratyphi A are estimated to account for 12-27 million febrile illness episodes worldwide annually. Determining the true burden of typhoidal Salmonellae infections is hindered by lack of population-based studies and adequate laboratory diagnostics.The Strategic Typhoid alliance across Africa and Asia study takes a systematic approach to measuring the age-stratified burden of clinical and subclinical disease caused by typhoidal Salmonellae infections at three high-incidence urban sites in Africa and Asia. We aim to explore the natural history of Salmonella transmission in endemic settings, addressing key uncertainties relating to the epidemiology of enteric fever identified through mathematical models, and enabling optimisation of vaccine strategies. METHODS/DESIGN: Using census-defined denominator populations of ≥100 000 individuals at sites in Malawi, Bangladesh and Nepal, the primary outcome is to characterise the burden of enteric fever in these populations over a 24-month period. During passive surveillance, clinical and household data, and laboratory samples will be collected from febrile individuals. In parallel, healthcare utilisation and water, sanitation and hygiene surveys will be performed to characterise healthcare-seeking behaviour and assess potential routes of transmission. The rates of both undiagnosed and subclinical exposure to typhoidal Salmonellae (seroincidence), identification of chronic carriage and population seroprevalence of typhoid infection will be assessed through age-stratified serosurveys performed at each site. Secondary attack rates will be estimated among household contacts of acute enteric fever cases and possible chronic carriers. ETHICS AND DISSEMINATION: This protocol has been ethically approved by the Oxford Tropical Research Ethics Committee, the icddr,b Institutional Review Board, the Malawian National Health Sciences Research Committee and College of Medicine Research Ethics Committee and Nepal Health Research Council. The study is being conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. Informed consent was obtained before study enrolment. Results will be submitted to international peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: ISRCTN 12131979. ETHICS REFERENCES: Oxford (Oxford Tropical Research Ethics Committee 39-15).Bangladesh (icddr,b Institutional Review Board PR-15119).Malawi (National Health Sciences Research Committee 15/5/1599).Nepal (Nepal Health Research Council 306/2015).
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    Frequent transmission of the Mycobacterium tuberculosis Beijing lineage and positive selection for the EsxW Beijing variant in Vietnam
    Holt, KE ; McAdam, P ; Phan, VKT ; Nguyen, TTT ; Dang, TMH ; Nguyen, NL ; Nguyen, HL ; Nguyen, TQN ; Hoang, TH ; Vu, TNH ; Thwaites, G ; Edwards, DJ ; Nath, AP ; Pham, K ; Ascher, DB ; Farrar, J ; Khor, CC ; Teo, YY ; Inouye, M ; Caws, M ; Dunstan, SJ (NATURE PUBLISHING GROUP, 2018-06)
    To examine the transmission dynamics of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients in Ho Chi Minh City, Vietnam, we sequenced the whole genomes of 1,635 isolates and compared these with 3,144 isolates from elsewhere. The data identify an underlying burden of disease caused by the endemic Mtb lineage 1 associated with the activation of long-term latent infection, and a threefold higher burden associated with the more recently introduced Beijing lineage and lineage 4 Mtb strains. We find that Beijing lineage Mtb is frequently transferred between Vietnam and other countries, and detect higher levels of transmission of Beijing lineage strains within this host population than the endemic lineage 1 Mtb. Screening for parallel evolution of Beijing lineage-associated SNPs in other Mtb lineages as a signal of positive selection, we identify an alteration in the ESX-5 type VII-secreted protein EsxW, which could potentially contribute to the enhanced transmission of Beijing lineage Mtb in Vietnamese and other host populations.
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    Empirical ways to identify novel Bedaquiline resistance mutations in AtpE
    Karmakar, M ; Rodrigues, CHM ; Holt, KE ; Dunstan, SJ ; Denholm, J ; Ascher, DB ; Mokrousov, I (PUBLIC LIBRARY SCIENCE, 2019-05-29)
    Clinical resistance against Bedaquiline, the first new anti-tuberculosis compound with a novel mechanism of action in over 40 years, has already been detected in Mycobacterium tuberculosis. As a new drug, however, there is currently insufficient clinical data to facilitate reliable and timely identification of genomic determinants of resistance. Here we investigate the structural basis for M. tuberculosis associated bedaquiline resistance in the drug target, AtpE. Together with the 9 previously identified resistance-associated variants in AtpE, 54 non-resistance-associated mutations were identified through comparisons of bedaquiline susceptibility across 23 different mycobacterial species. Computational analysis of the structural and functional consequences of these variants revealed that resistance associated variants were mainly localized at the drug binding site, disrupting key interactions with bedaquiline leading to reduced binding affinity. This was used to train a supervised predictive algorithm, which accurately identified likely resistance mutations (93.3% accuracy). Application of this model to circulating variants present in the Asia-Pacific region suggests that current circulating variants are likely to be susceptible to bedaquiline. We have made this model freely available through a user-friendly web interface called SUSPECT-BDQ, StrUctural Susceptibility PrEdiCTion for bedaquiline (http://biosig.unimelb.edu.au/suspect_bdq/). This tool could be useful for the rapid characterization of novel clinical variants, to help guide the effective use of bedaquiline, and to minimize the spread of clinical resistance.