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    High prevalence of PI resistance in patients failing second-line ART in Vietnam
    Vu, PT ; Vo, MQ ; Day, JN ; Nguyen, TC ; Shikuma, CM ; Farrar, J ; Nguyen, VVC ; Thwaites, GE ; Dunstan, SJ ; Thuy, L (OXFORD UNIV PRESS, 2016-03)
    BACKGROUND: There are limited data from resource-limited settings on antiretroviral resistance mutations that develop in patients failing second-line PI ART. METHODS: We performed a cross-sectional virological assessment of adults on second-line ART for ≥6 months between November 2006 and December 2011, followed by a prospective follow-up over 2 years of patients with virological failure (VF) at the Hospital for Tropical Diseases, Vietnam. VF was defined as HIV RNA concentrations ≥1000 copies/mL. Resistance mutations were identified by population sequencing of the pol gene and interpreted using the 2014 IAS-USA mutation list and the Stanford algorithm. Logistic regression modelling was performed to identify predictors of VF. RESULTS: Two hundred and thirty-one patients were enrolled in the study. The median age was 32 years; 81.0% were male, 95.7% were on a lopinavir/ritonavir-containing regimen and 22 (9.5%) patients had VF. Of the patients with VF, 14 (64%) carried at least one major protease mutation [median: 2 (IQR: 1-3)]; 13 (59%) had multiple protease mutations conferring intermediate- to high-level resistance to lopinavir/ritonavir. Mutations conferring cross-resistance to etravirine, rilpivirine, tipranavir and darunavir were identified in 55%, 55%, 45% and 27% of patients, respectively. Higher viral load, adherence <95% and previous indinavir use were independent predictors of VF. The 2 year outcomes of the patients maintained on lopinavir/ritonavir included: death, 7 (35%); worsening virological/immunological control, 6 (30%); and virological re-suppression, 5 (25%). Two patients were switched to raltegravir and darunavir/ritonavir with good HIV control. CONCLUSIONS: High-prevalence PI resistance was associated with previous indinavir exposure. Darunavir plus an integrase inhibitor and lamivudine might be a promising third-line regimen in Vietnam.
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    The variant call format and VCFtools
    Danecek, P ; Auton, A ; Abecasis, G ; Albers, CA ; Banks, E ; DePristo, MA ; Handsaker, RE ; Lunter, G ; Marth, GT ; Sherry, ST ; McVean, G ; Durbin, R (OXFORD UNIV PRESS, 2011-08-01)
    SUMMARY: The variant call format (VCF) is a generic format for storing DNA polymorphism data such as SNPs, insertions, deletions and structural variants, together with rich annotations. VCF is usually stored in a compressed manner and can be indexed for fast data retrieval of variants from a range of positions on the reference genome. The format was developed for the 1000 Genomes Project, and has also been adopted by other projects such as UK10K, dbSNP and the NHLBI Exome Project. VCFtools is a software suite that implements various utilities for processing VCF files, including validation, merging, comparing and also provides a general Perl API. AVAILABILITY: http://vcftools.sourceforge.net
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    Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia
    Clarke, GM ; Rockett, K ; Kivinen, K ; Hubbart, C ; Jeffreys, AE ; Rowlands, K ; Jallow, M ; Conway, DJ ; Bojang, KA ; Pinder, M ; Usen, S ; Sisay-Joof, F ; Sirugo, G ; Toure, O ; Thera, MA ; Konate, S ; Sissoko, S ; Niangaly, A ; Poudiougou, B ; Mangano, VD ; Bougouma, EC ; Sirima, SB ; Modioano, D ; Amenga-Etego, LN ; Ghansah, A ; Koram, KA ; Wilson, MD ; Enimil, A ; Evans, J ; Amodu, OK ; Olaniyan, S ; Apinjoh, T ; Mugri, R ; Ndi, A ; Ndila, CM ; Uyoga, S ; Macharia, A ; Peshu, N ; Williams, TN ; Manjurano, A ; Sepulveda, N ; Clark, TG ; Riley, E ; Drakeley, C ; Reyburn, H ; Nyirongo, V ; Kachala, D ; Molyneux, M ; Dunstan, SJ ; Nguyen, HP ; Nguyen, NQ ; Cao, QT ; Tran, TH ; Manning, L ; Laman, M ; Siba, P ; Karunajeewa, H ; Allen, S ; Allen, A ; Davis, TME ; Michon, P ; Mueller, I ; Molloy, SF ; Campino, S ; Kerasidou, A ; Cornelius, VJ ; Hart, L ; Shah, SS ; Band, G ; Spencer, CCA ; Agbenyega, T ; Achidi, E ; Doumbo, OK ; Farrar, J ; Marsh, K ; Taylor, T ; Kwaitkowski, DP (ELIFE SCIENCES PUBLICATIONS LTD, 2017-01-09)
    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against Plasmodium falciparum malaria, but the precise nature of the protective effecthas proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of P. falciparum infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual's level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of P. falciparum infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations.
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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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    Sources of Multidrug Resistance in Patients With Previous Isoniazid-Resistant Tuberculosis Identified Using Whole Genome Sequencing: A Longitudinal Cohort Study
    Srinivasan, V ; Ha, VTN ; Vinh, DN ; Thai, PVK ; Ha, DTM ; Lan, NH ; Hai, HT ; Walker, TM ; Thu, DDA ; Dunstan, SJ ; Thwaites, GE ; Ashton, PM ; Caws, M ; Thuong, NTT (OXFORD UNIV PRESS INC, 2020-11-15)
    BACKGROUND: Meta-analysis of patients with isoniazid-resistant tuberculosis (TB) given standard first-line anti-TB treatment indicated an increased risk of multidrug-resistant TB (MDR-TB) emerging (8%), compared to drug-sensitive TB (0.3%). Here we use whole genome sequencing (WGS) to investigate whether treatment of patients with preexisting isoniazid-resistant disease with first-line anti-TB therapy risks selecting for rifampicin resistance, and hence MDR-TB. METHODS: Patients with isoniazid-resistant pulmonary TB were recruited and followed up for 24 months. Drug susceptibility testing was performed by microscopic observation drug susceptibility assay, mycobacterial growth indicator tube, and by WGS on isolates at first presentation and in the case of re-presentation. Where MDR-TB was diagnosed, WGS was used to determine the genomic relatedness between initial and subsequent isolates. De novo emergence of MDR-TB was assumed where the genomic distance was 5 or fewer single-nucleotide polymorphisms (SNPs), whereas reinfection with a different MDR-TB strain was assumed where the distance was 10 or more SNPs. RESULTS: Two hundred thirty-nine patients with isoniazid-resistant pulmonary TB were recruited. Fourteen (14/239 [5.9%]) patients were diagnosed with a second episode of TB that was multidrug resistant. Six (6/239 [2.5%]) were identified as having evolved MDR-TB de novo and 6 as having been reinfected with a different strain. In 2 cases, the genomic distance was between 5 and 10 SNPs and therefore indeterminate. CONCLUSIONS: In isoniazid-resistant TB, de novo emergence and reinfection of MDR-TB strains equally contributed to MDR development. Early diagnosis and optimal treatment of isoniazid-resistant TB are urgently needed to avert the de novo emergence of MDR-TB during treatment.
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    Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study
    Ndila, CM ; Uyoga, S ; Macharia, AW ; Nyutu, G ; Peshu, N ; Ojal, J ; Shebe, M ; Awuondo, KO ; Mturi, N ; Tsofa, B ; Sepulveda, N ; Clark, TG ; Band, G ; Clarke, G ; Rowlands, K ; Hubbart, C ; Jeffreys, A ; Kariuki, S ; Marsh, K ; Mackinnon, M ; Maitland, K ; Kwiatkowski, DP ; Rockett, KA ; Williams, TN (ELSEVIER SCI LTD, 2018-08)
    BACKGROUND: Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms-many related to the structure or function of red blood cells-and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress. METHODS: We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria. FINDINGS: Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11-0·20; p=2·61 × 10-58), blood group O (0·74, 0·66-0·82; p=6·26 × 10-8), and -α3·7-thalassaemia (0·83, 0·76-0·90; p=2·06 × 10-6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63-0·92; p=0·001) and FREM3 (0·64, 0·53-0·79; p=3·18 × 10-14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49-0·68; p=3·22 × 10-11), as was homozygosity (0·26, 0·11-0·62; p=0·002). INTERPRETATION: Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health. FUNDING: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.
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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.
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    Bacterial risk factors for treatment failure and relapse among patients with isoniazid resistant tuberculosis
    Phan, VKT ; Dang, TMH ; Nguyen, TH ; Day, J ; Dunstan, S ; Nguyen, TQN ; Vo, SK ; Nguyen, HL ; Nguyen, HD ; Nguyen, TNL ; Nguyen, TT ; Nguyen, NL ; Pham, TTL ; Nguyen, TH ; Dao, CD ; Nguyen, TKT ; Nguyen, VH ; Nguyen, VN ; Truong, ND ; Hoang, QM ; Nguyen, VT ; Farrar, J ; Caws, M (BIOMED CENTRAL LTD, 2018-03-06)
    BACKGROUND: Drug resistant tuberculosis (TB) is increasing in prevalence worldwide. Treatment failure and relapse is known to be high for patients with isoniazid resistant TB treated with standard first line regimens. However, risk factors for unfavourable outcomes and the optimal treatment regimen for isoniazid resistant TB are unknown. This cohort study was conducted when Vietnam used the eight month first line treatment regimen and examined risk factors for failure/relapse among patients with isoniazid resistant TB. METHODS: Between December 2008 and June 2011 2090 consecutive HIV-negative adults (≥18 years of age) with new smear positive pulmonary TB presenting at participating district TB units in Ho Chi Minh City were recruited. Participants with isoniazid resistant TB identified by Microscopic Observation Drug Susceptibility (MODS) had extended follow-up for 2 years with mycobacterial culture to test for relapse. MGIT drug susceptibility testing confirmed 239 participants with isoniazid resistant, rifampicin susceptible TB. Bacterial and demographic factors were analysed for association with treatment failure and relapse. RESULTS: Using only routine programmatic sputum smear microscopy for assessment, (months 2, 5 and 8) 30/239 (12.6%) had an unfavourable outcome by WHO criteria. Thirty-nine patients were additionally detected with unfavourable outcomes during 2 year follow up, giving a total of 69/239 (28.9%) of isoniazid (INH) resistant cases with unfavourable outcome by 2 years of follow-up. Beijing lineage was the only factor significantly associated with unfavourable outcome among INH-resistant TB cases during 2 years of follow-up. (adjusted OR = 3.16 [1.54-6.47], P = 0.002). CONCLUSION: One third of isoniazid resistant TB cases suffered failure/relapse within 2 years under the old eight month regimen. Over half of these cases were not identified by standard WHO recommended treatment monitoring. Intensified research on early identification and optimal regimens for isoniazid resistant TB is needed. Infection with Beijing genotype of TB is a significant risk factor for bacterial persistence on treatment resulting in failure/relapse within 2 years. The underlying mechanism of increased tolerance for standard drug regimens in Beijing genotype strains remains unknown.
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    Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol
    Alvarez, MI ; Glover, LC ; Luo, P ; Wang, L ; Theusch, E ; Oehlers, SH ; Walton, EM ; Trinh, TBT ; Kuang, Y-L ; Rotter, JI ; McClean, CM ; Nguyen, TC ; Medina, MW ; Tobin, DM ; Dunstan, SJ ; Ko, DC (NATL ACAD SCIENCES, 2017-09-12)
    Risk, severity, and outcome of infection depend on the interplay of pathogen virulence and host susceptibility. Systematic identification of genetic susceptibility to infection is being undertaken through genome-wide association studies, but how to expeditiously move from genetic differences to functional mechanisms is unclear. Here, we use genetic association of molecular, cellular, and human disease traits and experimental validation to demonstrate that genetic variation affects expression of VAC14, a phosphoinositide-regulating protein, to influence susceptibility to Salmonella enterica serovar Typhi (S Typhi) infection. Decreased VAC14 expression increased plasma membrane cholesterol, facilitating Salmonella docking and invasion. This increased susceptibility at the cellular level manifests as increased susceptibility to typhoid fever in a Vietnamese population. Furthermore, treating zebrafish with a cholesterol-lowering agent, ezetimibe, reduced susceptibility to S Typhi. Thus, coupling multiple genetic association studies with mechanistic dissection revealed how VAC14 regulates Salmonella invasion and typhoid fever susceptibility and may open doors to new prophylactic/therapeutic approaches.