register interest

Professor Stephen Baker

Research Area: Global Health
Technology Exchange: Bioinformatics
Scientific Themes: Tropical Medicine & Global Health and Tropical Medicine & Global Health
Keywords: Enteric infections

Stephen Baker is a Wellcome Trust/Royal Society funded molecular microbiologist based at the Wellcome Trust Major overseas programme (WT-MOP) in Ho Chi Minh City, Vietnam. He has been at the WT-MOP since November 2007 and is the head of the enteric infections research group. He holds an Oxford University research lecturer’s position. His group studies the microbiology, genetics, epidemiology and treatment of enteric infections in developing countries. Focal pathogens include Norovirus, Shigella spp. and Salmonella Typhi, the causative agents of diarrhea, dysentery and typhoid fever, respectively.

There are no collaborations listed for this principal investigator.

Darton TC, Meiring JE, Tonks S, Khan MA, Khanam F, Shakya M, Thindwa D, Baker S, Basnyat B, Clemens JD et al. 2017. 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. BMJ Open, 7 (6), pp. e016283. | Show Abstract | Read more

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).

Campbell JI, Huong Lan NP, Phuong PM, Buu Chau L, Duc TP, Guzmán-Verri C, Ruiz-Villalobos N, Thi Minh TP, Muñoz Álvaro PM, Moreno E et al. 2017. Human Brucella melitensis infections in southern Vietnam. Clin Microbiol Infect, | Read more

Day JN, Qihui S, Thanh LT, Trieu PH, Van AD, Thu NH, Chau TTH, Lan NPH, Chau NVV, Ashton PM et al. 2017. Comparative genomics of Cryptococcus neoformans var. grubii associated with meningitis in HIV infected and uninfected patients in Vietnam. PLoS Negl Trop Dis, 11 (6), pp. e0005628. | Show Abstract | Read more

The vast burden of cryptococcal meningitis occurs in immunosuppressed patients, driven by HIV, and is caused by Cryptococcus neoformans var. grubii. We previously reported cryptococcal meningitis in Vietnam arising atypically in HIV uninfected, apparently immunocompetent patients, caused by a single amplified fragment length polymorphism (AFLP) cluster of C. neoformans var. grubii (VNIγ). This variant was less common in HIV infected individuals; it remains unclear why this lineage is associated with apparently immunocompetent patients. To study this host tropism we aimed to further our understanding of clinical phenotype and genomic variation within Vietnamese C. neoformans var. grubii. After performing MLST on C. neoformans clinical isolates we identified 14 sequence types (STs); ST5 correlated with the VNIγ cluster. We next compared clinical phenotype by lineage and found HIV infected patients with cryptococcal meningitis caused by ST5 organisms were significantly more likely to have lymphadenopathy (11% vs. 4%, p = 0.05 Fisher's exact test) and higher blood lymphocyte count (median 0.76 versus 0.55 X109 cells/L, p = 0.001, Kruskal-Wallis test). Furthermore, survivors of ST5 infections had evidence of worse disability outcomes at 70 days (72.7% (40/55) in ST5 infections versus 57.1% (52/91) non-ST5 infections (OR 2.11, 95%CI 1.01 to 4.41), p = 0.046). To further investigate the relationship between strain and disease phenotype we performed genome sequencing on eight Vietnamese C. neoformans var. grubii. Eight genome assemblies exhibited >99% nucleotide sequence identity and we identified 165 kbp of lineage specific to Vietnamese isolates. ST5 genomes harbored several strain specific regions, incorporating 19 annotated coding sequences and eight hypothetical proteins. These regions included a phenolic acid decarboxylase, a DEAD-box ATP-dependent RNA helicase 26, oxoprolinases, a taurine catabolism dioxygenase, a zinc finger protein, membrane transport proteins and various drug transporters. Our work outlines the complexity of genomic pathogenicity in cryptococcal infections and identifies a number of gene candidates that may aid the disaggregation of the pathways associated with the pathogenesis of Cryptococcus neoformans var. grubii.

Berto A, Pham HA, Thao TTN, Vy NHT, Caddy SL, Hiraide R, Tue NT, Goodfellow I, Carrique-Mas JJ, Thwaites GE et al. 2017. Hepatitis E in southern Vietnam: Seroepidemiology in humans and molecular epidemiology in pigs. Zoonoses Public Health, | Show Abstract | Read more

Viral pathogens account for a significant proportion of the burden of emerging infectious diseases in humans. The Wellcome Trust-Vietnamese Initiative on Zoonotic Infections (WT-VIZIONS) is aiming to understand the circulation of viral zoonotic pathogens in animals that pose a potential risk to human health. Evidence suggests that human exposure and infections with hepatitis E virus (HEV) genotypes (GT) 3 and 4 results from zoonotic transmission. Hypothesising that HEV GT3 and GT4 are circulating in the Vietnamese pig population and can be transmitted to humans, we aimed to estimate the seroprevalence of HEV exposure in a population of farmers and the general population. We additionally performed sequence analysis of HEV in pig populations in the same region to address knowledge gaps regarding HEV circulation and to evaluate if pigs were a potential source of HEV exposure. We found a high prevalence of HEV GT3 viral RNA in pigs (19.1% in faecal samples and 8.2% in rectal swabs) and a high HEV seroprevalence in pig farmers (16.0%) and a hospital-attending population (31.7%) in southern Vietnam. The hospital population was recruited as a general-population proxy even though this particular population subgroup may introduce bias. The detection of HEV RNA in pigs indicates that HEV may be a zoonotic disease risk in this location, although a larger sample size is required to infer an association between HEV positivity in pigs and seroprevalence in humans.

Tran Vu Thieu N, Trinh Van T, Tran Tuan A, Klemm EJ, Nguyen Ngoc Minh C, Voong Vinh P, Pham Thanh D, Ho Ngoc Dan T, Pham Duc T, Langat P et al. 2017. An evaluation of purified Salmonella Typhi protein antigens for the serological diagnosis of acute typhoid fever. J Infect, 75 (2), pp. 104-114. | Show Abstract | Read more

OBJECTIVES: The diagnosis of typhoid fever is a challenge. Aiming to develop a typhoid diagnostic we measured antibody responses against Salmonella Typhi (S. Typhi) protein antigens and the Vi polysaccharide in a cohort of Bangladeshi febrile patients. METHODS: IgM against 12 purified antigens and the Vi polysaccharide was measured by ELISA in plasma from patients with confirmed typhoid fever (n = 32), other confirmed infections (n = 17), and healthy controls (n = 40). ELISAs with the most specific antigens were performed on plasma from 243 patients with undiagnosed febrile disease. RESULTS: IgM against the S. Typhi protein antigens correlated with each other (rho > 0.8), but not against Vi (rho < 0.6). Typhoid patients exhibited higher IgM against 11/12 protein antigens and Vi than healthy controls and those with other infections. Vi, PilL, and CdtB exhibited the greatest sensitivity and specificity. Specificity and sensitivity was improved when Vi was combined with a protein antigen, generating sensitivities and specificities of 0.80 and >0.85, respectively. Applying a dynamic cut-off to patients with undiagnosed febrile disease suggested that 34-58% had an IgM response indicative of typhoid. CONCLUSIONS: We evaluated the diagnostic potential of several S. Typhi antigens; our assays give good sensitivity and specificity, but require further assessment in differing patient populations.

Näsström E, Parry CM, Vu Thieu NT, Maude RR, de Jong HK, Fukushima M, Rzhepishevska O, Marks F, Panzner U, Im J et al. 2017. Reproducible diagnostic metabolites in plasma from typhoid fever patients in Asia and Africa. Elife, 6 | Show Abstract | Read more

Salmonella Typhi is the causative agent of typhoid. Typhoid is diagnosed by blood culture, a method that lacks sensitivity, portability and speed. We have previously shown that specific metabolomic profiles can be detected in the blood of typhoid patients from Nepal (Näsström et al., 2014). Here, we performed mass spectrometry on plasma from Bangladeshi and Senegalese patients with culture confirmed typhoid fever, clinically suspected typhoid, and other febrile diseases including malaria. After applying supervised pattern recognition modelling, we could significantly distinguish metabolite profiles in plasma from the culture confirmed typhoid patients. After comparing the direction of change and degree of multivariate significance, we identified 24 metabolites that were consistently up- or down regulated in a further Bangladeshi/Senegalese validation cohort, and the Nepali cohort from our previous work. We have identified and validated a metabolite panel that can distinguish typhoid from other febrile diseases, providing a new approach for typhoid diagnostics.

Saad NJ, Bowles CC, Grenfell BT, Basnyat B, Arjyal A, Dongol S, Karkey A, Baker S, Pitzer VE. 2017. The impact of migration and antimicrobial resistance on the transmission dynamics of typhoid fever in Kathmandu, Nepal: A mathematical modelling study. PLoS Negl Trop Dis, 11 (5), pp. e0005547. | Show Abstract | Read more

BACKGROUND: A substantial proportion of the global burden of typhoid fever occurs in South Asia. Kathmandu, Nepal experienced a substantial increase in the number of typhoid fever cases (caused by Salmonella Typhi) between 2000 and 2003, which subsequently declined but to a higher endemic level than in 2000. This epidemic of S. Typhi coincided with an increase in organisms with reduced susceptibility against fluoroquinolones, the emergence of S. Typhi H58, and an increase in the migratory population in Kathmandu. METHODS: We devised a mathematical model to investigate the potential epidemic drivers of typhoid in Kathmandu and fit this model to weekly data of S. Typhi cases between April 1997 and June 2011 and the age distribution of S. Typhi cases. We used this model to determine if the typhoid epidemic in Kathmandu was driven by heightened migration, the emergence of organisms with reduced susceptibility against fluoroquinolones or a combination of these factors. RESULTS: Models allowing for the migration of susceptible individuals into Kathmandu alone or in combination with the emergence of S. Typhi with reduced susceptibility against fluoroquinolones provided a good fit for the data. The emergence of organisms with reduced susceptibility against fluoroquinolones organisms alone, either through an increase in disease duration or increased transmission, did not fully explain the pattern of S. Typhi infections. CONCLUSIONS: Our analysis is consistent with the hypothesis that the increase in typhoid fever in Kathmandu was associated with the migration of susceptible individuals into the city and aided by the emergence of reduced susceptibility against fluoroquinolones. These data support identifying and targeting migrant populations with typhoid immunization programmes to prevent transmission and disease.

Dung TTN, Duy PT, Sessions OM, Sangumathi UK, Phat VV, Tam PTT, To NTN, Phuc TM, Hong Chau TT, Chau NNM et al. 2017. A universal genome sequencing method for rotavirus A from human fecal samples which identifies segment reassortment and multi-genotype mixed infection. BMC Genomics, 18 (1), pp. 324. | Show Abstract | Read more

BACKGROUND: Genomic characterization of rotavirus (RoV) has not been adopted at large-scale due to the complexity of obtaining sequences for all 11 segments, particularly when feces are used as starting material. METHODS: To overcome these limitations, we developed a novel RoV capture and genome sequencing method combining commercial enzyme immunoassay plates and a set of routinely used reagents. RESULTS: Our approach had a 100% success rate, producing >90% genome coverage for diverse RoV present in fecal samples (Ct < 30). CONCLUSIONS: This method provides a novel, reproducible and comparatively simple approach for genomic RoV characterization and could be scaled-up for use in global RoV surveillance systems. TRIAL REGISTRATION (PROSPECTIVELY REGISTERED): Current Controlled Trials ISRCTN88101063 . Date of registration: 14/06/2012.

Berto A, Anh PH, Carrique-Mas JJ, Simmonds P, Van Cuong N, Tue NT, Van Dung N, Woolhouse ME, Smith I, Marsh GA et al. 2017. Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam. Zoonoses Public Health, | Show Abstract | Read more

Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross-species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health.

Berto A, Day J, Van Vinh Chau N, Thwaites GE, My NN, Baker S, Darton TC. 2017. Current challenges and possible solutions to improve access to care and treatment for hepatitis C infection in Vietnam: a systematic review. BMC Infect Dis, 17 (1), pp. 260. | Show Abstract | Read more

BACKGROUND: Hepatitis C infection is a major public health concern in low- and middle-income countries where an estimated 71.1 million individuals are living with chronic infection. The World Health Organization (WHO) has recently released new guidance for hepatitis C virus (HCV) treatment programs, which include improving the access to new direct-acting antiviral agents. In Vietnam, a highly populated middle-income country, the seroprevalence of HCV infection is approximately 4% and multiple genotypes co-circulate in the general population. Here we review what is currently known regarding the epidemiology of HCV in Vietnam and outline options for reducing the significant burden of morbidity and mortality in our setting. METHODS: We performed a systematic review of the currently available literature to evaluate what has been achieved to date with efforts to control HCV infection in Vietnam. RESULTS: This search retrieved few publications specific to Vietnam indicating a significant gap in baseline epidemiological and public health data. Key knowledge gaps identified included an understanding of the prevalence in specific high-risk groups, characterization of circulating HCV genotypes in the population and likely response to treatment, and the extent to which HCV treatment is available, accessed and utilized. CONCLUSIONS: We conclude that there is an urgent need to perform up to date assessments of HCV disease burden in Vietnam, especially in high-risk groups, in whom incidence is high and cross infection with multiple genotypes is likely to be frequent. Coordinating renewed surveillance measures with forthcoming HCV treatment studies should initiate the traction required to achieve the WHO goal of eliminating HCV as a public health threat by 2030, at least in this region.

Darton TC, Zhou L, Blohmke CJ, Jones C, Waddington CS, Baker S, Pollard AJ. 2017. Blood culture-PCR to optimise typhoid fever diagnosis after controlled human infection identifies frequent asymptomatic cases and evidence of primary bacteraemia. J Infect, 74 (4), pp. 358-366. | Show Abstract | Read more

BACKGROUND: Improved diagnostics for typhoid are needed; a typhoid controlled human infection model may accelerate their development and translation. Here, we evaluated a blood culture-PCR assay for detecting infection after controlled human infection with S. Typhi and compared test performance with optimally performed blood cultures. METHODOLOGY/PRINCIPAL FINDINGS: Culture-PCR amplification of blood samples was performed alongside daily blood culture in 41 participants undergoing typhoid challenge. Study endpoints for typhoid diagnosis (TD) were fever and/or bacteraemia. Overall, 24/41 (59%) participants reached TD, of whom 21/24 (86%) had ≥1 positive blood culture (53/674, 7.9% of all cultures) or 18/24 (75%) had ≥1 positive culture-PCR assay result (57/684, 8.3%). A further five non-bacteraemic participants produced culture-PCR amplicons indicating infection; overall sensitivity/specificity of the assay compared to the study endpoints were 70%/65%. We found no significant difference between blood culture and culture-PCR methods in ability to identify cases (12 mismatching pairs, p = 0.77, binomial test). Clinical and stool culture metadata demonstrated that additional culture-PCR amplification positive individuals likely represented true cases missed by blood culture, suggesting the overall attack rate may be 30/41 (73%) rather than 24/41 (59%). Several participants had positive culture-PCR results soon after ingesting challenge providing new evidence for occurrence of an early primary bacteraemia. CONCLUSIONS/SIGNIFICANCE: Overall the culture-PCR assay performed well, identifying extra typhoid cases compared with routine blood culture alone. Despite limitations to widespread field-use, the benefits of increased diagnostic yield, reduced blood volume and faster turn-around-time, suggest that this assay could enhance laboratory typhoid diagnostics in research applications and high-incidence settings.

Marks F, von Kalckreuth V, Aaby P, Adu-Sarkodie Y, El Tayeb MA, Ali M, Aseffa A, Baker S, Biggs HM, Bjerregaard-Andersen M et al. 2017. Incidence of invasive salmonella disease in sub-Saharan Africa: a multicentre population-based surveillance study. Lancet Glob Health, 5 (3), pp. e310-e323. | Show Abstract | Read more

BACKGROUND: Available incidence data for invasive salmonella disease in sub-Saharan Africa are scarce. Standardised, multicountry data are required to better understand the nature and burden of disease in Africa. We aimed to measure the adjusted incidence estimates of typhoid fever and invasive non-typhoidal salmonella (iNTS) disease in sub-Saharan Africa, and the antimicrobial susceptibility profiles of the causative agents. METHODS: We established a systematic, standardised surveillance of blood culture-based febrile illness in 13 African sentinel sites with previous reports of typhoid fever: Burkina Faso (two sites), Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar (two sites), Senegal, South Africa, Sudan, and Tanzania (two sites). We used census data and health-care records to define study catchment areas and populations. Eligible participants were either inpatients or outpatients who resided within the catchment area and presented with tympanic (≥38·0°C) or axillary temperature (≥37·5°C). Inpatients with a reported history of fever for 72 h or longer were excluded. We also implemented a health-care utilisation survey in a sample of households randomly selected from each study area to investigate health-seeking behaviour in cases of self-reported fever lasting less than 3 days. Typhoid fever and iNTS disease incidences were corrected for health-care-seeking behaviour and recruitment. FINDINGS: Between March 1, 2010, and Jan 31, 2014, 135 Salmonella enterica serotype Typhi (S Typhi) and 94 iNTS isolates were cultured from the blood of 13 431 febrile patients. Salmonella spp accounted for 33% or more of all bacterial pathogens at nine sites. The adjusted incidence rate (AIR) of S Typhi per 100 000 person-years of observation ranged from 0 (95% CI 0-0) in Sudan to 383 (274-535) at one site in Burkina Faso; the AIR of iNTS ranged from 0 in Sudan, Ethiopia, Madagascar (Isotry site), and South Africa to 237 (178-316) at the second site in Burkina Faso. The AIR of iNTS and typhoid fever in individuals younger than 15 years old was typically higher than in those aged 15 years or older. Multidrug-resistant S Typhi was isolated in Ghana, Kenya, and Tanzania (both sites combined), and multidrug-resistant iNTS was isolated in Burkina Faso (both sites combined), Ghana, Kenya, and Guinea-Bissau. INTERPRETATION: Typhoid fever and iNTS disease are major causes of invasive bacterial febrile illness in the sampled locations, most commonly affecting children in both low and high population density settings. The development of iNTS vaccines and the introduction of S Typhi conjugate vaccines should be considered for high-incidence settings, such as those identified in this study. FUNDING: Bill & Melinda Gates Foundation.

Thompson CN, Karkey A, Dongol S, Arjyal A, Wolbers M, Darton T, Farrar JJ, Thwaites GE, Dolecek C, Basnyat B, Baker S. 2017. Treatment Response in Enteric Fever in an Era of Increasing Antimicrobial Resistance: An Individual Patient Data Analysis of 2092 Participants Enrolled into 4 Randomized, Controlled Trials in Nepal. Clin Infect Dis, 64 (11), pp. 1522-1531. | Show Abstract | Read more

Background.: Enteric fever, caused by Salmonella Typhi and Salmonella Paratyphi A, is the leading cause of bacterial febrile disease in South Asia. Methods.: Individual data from 2092 patients with enteric fever randomized into 4 trials in Kathmandu, Nepal, were pooled. All trials compared gatifloxacin with 1 of the following comparator drugs: cefixime, chloramphenicol, ofloxacin, or ceftriaxone. Treatment outcomes were evaluated according to antimicrobial if S. Typhi/Paratyphi were isolated from blood. We additionally investigated the impact of changing bacterial antimicrobial susceptibility on outcome. Results.: Overall, 855 (41%) patients had either S. Typhi (n = 581, 28%) or S. Paratyphi A (n = 274, 13%) cultured from blood. There were 139 (6.6%) treatment failures with 1 death. Except for the last trial with ceftriaxone, the fluoroquinolone gatifloxacin was associated with equivalent or better fever clearance times and lower treatment failure rates in comparison to all other antimicrobials. However, we additionally found that the minimum inhibitory concentrations (MICs) against fluoroquinolones have risen significantly since 2005 and were associated with increasing fever clearance times. Notably, all organisms were susceptible to ceftriaxone throughout the study period (2005-2014), and the MICs against azithromycin declined, confirming the utility of these alternative drugs for enteric fever treatment. Conclusion.: The World Health Organization and local government health ministries in South Asia still recommend fluoroquinolones for enteric fever. This policy should change based on the evidence provided here. Rapid diagnostics are urgently required given the large numbers of suspected enteric fever patients with a negative culture.

Kintz E, Heiss C, Black I, Donohue N, Brown N, Davies MR, Azadi P, Baker S, Kaye PM, van der Woude M. 2017. Salmonella enterica Serovar Typhi Lipopolysaccharide O-Antigen Modification Impact on Serum Resistance and Antibody Recognition. Infect Immun, 85 (4), pp. e01021-16-e01021-16. | Show Abstract | Read more

Salmonella enterica serovar Typhi is a human-restricted Gram-negative bacterial pathogen responsible for causing an estimated 27 million cases of typhoid fever annually, leading to 217,000 deaths, and current vaccines do not offer full protection. The O-antigen side chain of the lipopolysaccharide is an immunodominant antigen, can define host-pathogen interactions, and is under consideration as a vaccine target for some Gram-negative species. The composition of the O-antigen can be modified by the activity of glycosyltransferase (gtr) operons acquired by horizontal gene transfer. Here we investigate the role of two gtr operons that we identified in the S Typhi genome. Strains were engineered to express specific gtr operons. Full chemical analysis of the O-antigens of these strains identified gtr-dependent glucosylation and acetylation. The glucosylated form of the O-antigen mediated enhanced survival in human serum and decreased complement binding. A single nucleotide deviation from an epigenetic phase variation signature sequence rendered the expression of this glucosylating gtr operon uniform in the population. In contrast, the expression of the acetylating gtrC gene is controlled by epigenetic phase variation. Acetylation did not affect serum survival, but phase variation can be an immune evasion mechanism, and thus, this modification may contribute to persistence in a host. In murine immunization studies, both O-antigen modifications were generally immunodominant. Our results emphasize that natural O-antigen modifications should be taken into consideration when assessing responses to vaccines, especially O-antigen-based vaccines, and that the Salmonellagtr repertoire may confound the protective efficacy of broad-ranging Salmonella lipopolysaccharide conjugate vaccines.

Dobinson HC, Gibani MM, Jones C, Thomaides-Brears HB, Voysey M, Darton TC, Waddington CS, Campbell D, Milligan I, Zhou L et al. 2017. Evaluation of the Clinical and Microbiological Response to Salmonella Paratyphi A Infection in the First Paratyphoid Human Challenge Model. Clin Infect Dis, 64 (8), pp. 1066-1073. | Show Abstract | Read more

Background.: To expedite the evaluation of vaccines against paratyphoid fever, we aimed to develop the first human challenge model of Salmonella enterica serovar Paratyphi A infection. Methods.: Two groups of 20 participants underwent oral challenge with S. Paratyphi A following sodium bicarbonate pretreatment at 1 of 2 dose levels (group 1: 1-5 × 103 colony-forming units [CFU] and group 2: 0.5-1 × 103 CFU). Participants were monitored in an outpatient setting with daily clinical review and collection of blood and stool cultures. Antibiotic treatment was started when prespecified diagnostic criteria were met (temperature ≥38°C for ≥12 hours and/or bacteremia) or at day 14 postchallenge. Results.: The primary study objective was achieved following challenge with 1-5 × 103 CFU (group 1), which resulted in an attack rate of 12 of 20 (60%). Compared with typhoid challenge, paratyphoid was notable for high rates of subclinical bacteremia (at this dose, 11/20 [55%]). Despite limited symptoms, bacteremia persisted for up to 96 hours after antibiotic treatment (median duration of bacteremia, 53 hours [interquartile range, 24-85 hours]). Shedding of S. Paratyphi A in stool typically preceded onset of bacteremia. Conclusions.: Challenge with S. Paratyphi A at a dose of 1-5 × 103 CFU was well tolerated and associated with an acceptable safety profile. The frequency and persistence of bacteremia in the absence of clinical symptoms was notable, and markedly different from that seen in previous typhoid challenge studies. We conclude that the paratyphoid challenge model is suitable for the assessment of vaccine efficacy using endpoints that include bacteremia and/or symptomatology. Clinical Trials Registration.: NCT02100397.

Baker S. 2017. Emerging infectious diseases in Asia Microbiology Today, 44 (1), pp. 26-29.

Dyson ZA, Thanh DP, Bodhidatta L, Mason CJ, Srijan A, Rabaa MA, Vinh PV, Thanh TH, Thwaites GE, Baker S, Holt KE. 2017. Whole Genome Sequence Analysis of Salmonella Typhi Isolated in Thailand before and after the Introduction of a National Immunization Program. PLoS Negl Trop Dis, 11 (1), pp. e0005274. | Show Abstract | Read more

Vaccines against Salmonella Typhi, the causative agent of typhoid fever, are commonly used by travellers, however, there are few examples of national immunization programs in endemic areas. There is therefore a paucity of data on the impact of typhoid immunization programs on localised populations of S. Typhi. Here we have used whole genome sequencing (WGS) to characterise 44 historical bacterial isolates collected before and after a national typhoid immunization program that was implemented in Thailand in 1977 in response to a large outbreak; the program was highly effective in reducing typhoid case numbers. Thai isolates were highly diverse, including 10 distinct phylogenetic lineages or genotypes. Novel prophage and plasmids were also detected, including examples that were previously only reported in Shigella sonnei and Escherichia coli. The majority of S. Typhi genotypes observed prior to the immunization program were not observed following it. Post-vaccine era isolates were more closely related to S. Typhi isolated from neighbouring countries than to earlier Thai isolates, providing no evidence for the local persistence of endemic S. Typhi following the national immunization program. Rather, later cases of typhoid appeared to be caused by the occasional importation of common genotypes from neighbouring Vietnam, Laos, and Cambodia. These data show the value of WGS in understanding the impacts of vaccination on pathogen populations and provide support for the proposal that large-scale typhoid immunization programs in endemic areas could result in lasting local disease elimination, although larger prospective studies are needed to test this directly.

Tuan Anh N, Nga TV, Tuan HM, Tuan NS, Y DM, Vinh Chau NV, Baker S, Duong HH. 2017. Molecular epidemiology and antimicrobial resistance phenotypes of Acinetobacter baumannii isolated from patients in three hospitals in southern Vietnam. J Med Microbiol, 66 (1), pp. 46-53. | Show Abstract | Read more

Multidrug resistance in the nosocomial pathogen Acinetobacter baumannii limits therapeutic options and impacts on clinical care. Resistance against carbapenems, a group of last-resort antimicrobials for treating multidrug-resistant (MDR) A. baumannii infections, is associated with the expression (and over-expression) of carbapenemases encoded by the blaOXA genes. The aim of this study was to determine the prevalence of antimicrobial-resistant A. baumannii associated with infection in three hospitals in southern Vietnam and to characterize the genetic determinants associated with resistance against carbapenems. We recovered a total of 160 A. baumannii isolates from clinical samples collected in three hospitals in southern Vietnam from 2012 to 2014. Antimicrobial resistance was common; 119/160 (74 %) of isolates were both MDR and extensively drug resistant (XDR). High-level imipenem resistance (>32 µg ml-1) was determined for 109/117 (91.6 %) of the XDR imipenem-nonsusceptible organisms, of which the majority (86.7 %) harboured the blaOXA-51 and blaOXA-23 genes associated with an ISAba1 element. Multiple-locus variable number tandem repeat analysis segregated the 160 A. baumannii into 107 different multiple-locus variable number tandem repeat analysis types, which described five major clusters. The biggest cluster was a clonal complex composed mainly of imipenem-resistant organisms that were isolated from all three of the study hospitals. Our study indicates a very high prevalence of MDR/XDR A. baumannii causing clinically significant infections in hospitals in southern Vietnam. These organisms commonly harboured the blaOXA-23 gene with ISAba1 and were carbapenem resistant; this resistance phenotype may explain their continued selection and ongoing transmission within the Vietnamese healthcare system.

Tuan Anh N, Nga TV, Tuan HM, Tuan NS, Y DM, Chau NV, Baker S, Duong HH. 2016. The molecular epidemiology and antimicrobial resistance phenotypes of Acinetobacter baumannii isolated from patients in three hospitals in southern Vietnam. J Med Microbiol, 66 (1), pp. 46-53. | Show Abstract | Read more

Multidrug resistance (MDR) in the nosocomial pathogen Acinetobacter baumannii limits therapeutic options and impacts on clinical care. Resistance against carbapenems, a group of last-resort antimicrobials for treating MDR A. baumannii infections, is associated with the expression (and over-expression) of carbapenemases encoded by the blaOXA genes. The aim of this study was to determine the prevalence of antimicrobial resistant A. baumannii associated with infection in three hospitals in southern Vietnam and to characterise the genetic determinants associated with resistance against carbapenems. We recovered a total of 160 A. baumannii isolates from clinical samples collected in three hospitals in southern Vietnam from 2012 to 2014. Antimicrobial resistance was common; 119/160 (74%) of isolates were both MDR and extensively drug resistant (XDR). High-level imipenem resistance (>32µg/ml) was determined for 109/117 (91.6%) of the XDR imipenem non-susceptible organisms, of which the majority (86.7%) harboured the blaOXA-51 and blaOXA-23 genes associated with an ISAba1 element. Multiple-locus variable number tandem repeat analysis (MLVA) segregated the 160 A. baumannii into 107 different MLVA types, which described five major clusters. The biggest cluster was a clonal complex comprised mainly of imipenem resistant organisms that were isolated from all three of the study hospitals. Our study indicates a very high prevalence of MDR/XDR A. baumannii causing clinically significant infections in hospitals in southern Vietnam. These organisms commonly harboured the blaOXA-23 gene with ISAba1 and were carbapenem resistant; this resistance phenotype may explain their continued selection and on-going transmission within the Vietnamese healthcare system.

Karki M, Pandit S, Baker S, Basnyat B. 2016. Cotrimoxazole treats fluoroquinolone-resistant Salmonella typhi H58 infection. BMJ Case Rep, 2016 pp. bcr2016217223-bcr2016217223. | Show Abstract | Read more

A woman aged 20 years presented with fever and no localising signs. She was treated with cotrimoxazole and the subsequent blood culture was positive for Salmonella typhi (S. typhi), which was resistant to fluoroquinolones but susceptible to cotrimoxazole. Genotyping identified an FQ-R subclade of H58 S. typhi Fever clearance time was 4 days after starting the antibiotics, and no relapses were noted on 2 months of follow-up. This inexpensive, well-known and easily available antimicrobial could be suitably redeployed for fluoroquinolone-resistant enteric fever in South Asia.

Maude RR, Ghose A, Samad R, de Jong HK, Fukushima M, Wijedoru L, Hassan MU, Hossain MA, Karim MR, Sayeed AA et al. 2016. A prospective study of the importance of enteric fever as a cause of non-malarial febrile illness in patients admitted to Chittagong Medical College Hospital, Bangladesh. BMC Infect Dis, 16 (1), pp. 567. | Show Abstract | Read more

BACKGROUND: Fever is a common cause of hospital admission in Bangladesh but causative agents, other than malaria, are not routinely investigated. Enteric fever is thought to be common. METHODS: Adults and children admitted to Chittagong Medical College Hospital with a temperature of ≥38.0 °C were investigated using a blood smear for malaria, a blood culture, real-time PCR to detect Salmonella Typhi, S. Paratyphi A and other pathogens in blood and CSF and an NS1 antigen dengue ELISA. RESULTS: We enrolled 300 febrile patients with a negative malaria smear between January and June 2012: 156 children (aged ≤15 years) and 144 adults with a median (interquartile range) age of 13 (5-31) years and median (IQR) illness duration before admission of five (2-8) days. Clinical enteric fever was diagnosed in 52 patients (17.3 %), lower respiratory tract infection in 48 (16.0 %), non-specific febrile illness in 48 (16.0 %), a CNS infection in 37 patients (12.3 %), urinary sepsis in 23 patients (7.7 %), an upper respiratory tract infection in 21 patients (7.0 %), and diarrhea or dysentery in 21 patients (7.0 %). Malaria was still suspected in seven patients despite a negative microscopy test. S. Typhi was detected in blood by culture or PCR in 34 (11.3 %) of patients. Of note Rickettsia typhi and Orientia tsutsugamushi were detected by PCR in two and one patient respectively. Twenty-nine (9 %) patients died during their hospital admission (15/160 (9.4 %) of children and 14/144 (9.7 %) adults). Two of 52 (3.8 %) patients with enteric fever, 5/48 (10.4 %) patients with lower respiratory tract infections, and 12/37 (32.4 %) patients with CNS infection died. CONCLUSION: Enteric fever was confirmed in 11.3 % of patients admitted to this hospital in Bangladesh with non-malaria fever. Lower respiratory tract and CNS infections were also common. CNS infections in this location merit more detailed study due to the high mortality.

Wong VK, Baker S, Connor TR, Pickard D, Page AJ, Dave J, Murphy N, Holliman R, Sefton A, Millar M et al. 2016. An extended genotyping framework for Salmonella enterica serovar Typhi, the cause of human typhoid. Nat Commun, 7 pp. 12827. | Show Abstract | Read more

The population of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, exhibits limited DNA sequence variation, which complicates efforts to rationally discriminate individual isolates. Here we utilize data from whole-genome sequences (WGS) of nearly 2,000 isolates sourced from over 60 countries to generate a robust genotyping scheme that is phylogenetically informative and compatible with a range of assays. These data show that, with the exception of the rapidly disseminating H58 subclade (now designated genotype 4.3.1), the global S. Typhi population is highly structured and includes dozens of subclades that display geographical restriction. The genotyping approach presented here can be used to interrogate local S. Typhi populations and help identify recent introductions of S. Typhi into new or previously endemic locations, providing information on their likely geographical source. This approach can be used to classify clinical isolates and provides a universal framework for further experimental investigations.

International Typhoid Consortium, Wong VK, Holt KE, Okoro C, Baker S, Pickard DJ, Marks F, Page AJ, Olanipekun G, Munir H et al. 2016. Molecular Surveillance Identifies Multiple Transmissions of Typhoid in West Africa. PLoS Negl Trop Dis, 10 (9), pp. e0004781. | Show Abstract | Read more

BACKGROUND: The burden of typhoid in sub-Saharan African (SSA) countries has been difficult to estimate, in part, due to suboptimal laboratory diagnostics. However, surveillance blood cultures at two sites in Nigeria have identified typhoid associated with Salmonella enterica serovar Typhi (S. Typhi) as an important cause of bacteremia in children. METHODS: A total of 128 S. Typhi isolates from these studies in Nigeria were whole-genome sequenced, and the resulting data was used to place these Nigerian isolates into a worldwide context based on their phylogeny and carriage of molecular determinants of antibiotic resistance. RESULTS: Several distinct S. Typhi genotypes were identified in Nigeria that were related to other clusters of S. Typhi isolates from north, west and central regions of Africa. The rapidly expanding S. Typhi clade 4.3.1 (H58) previously associated with multiple antimicrobial resistances in Asia and in east, central and southern Africa, was not detected in this study. However, antimicrobial resistance was common amongst the Nigerian isolates and was associated with several plasmids, including the IncHI1 plasmid commonly associated with S. Typhi. CONCLUSIONS: These data indicate that typhoid in Nigeria was established through multiple independent introductions into the country, with evidence of regional spread. MDR typhoid appears to be evolving independently of the haplotype H58 found in other typhoid endemic countries. This study highlights an urgent need for routine surveillance to monitor the epidemiology of typhoid and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions to reduce the substantial morbidity and mortality of typhoid.

Phu Huong Lan N, Le Thi Phuong T, Nguyen Huu H, Thuy L, Mather AE, Park SE, Marks F, Thwaites GE, Van Vinh Chau N, Thompson CN, Baker S. 2016. Invasive Non-typhoidal Salmonella Infections in Asia: Clinical Observations, Disease Outcome and Dominant Serovars from an Infectious Disease Hospital in Vietnam. PLoS Negl Trop Dis, 10 (8), pp. e0004857. | Show Abstract | Read more

Invasive non-typhoidal Salmonella (iNTS) infections are now a well-described cause of morbidity and mortality in children and HIV-infected adults in sub-Saharan Africa. In contrast, the epidemiology and clinical manifestations of iNTS disease in Asia are not well documented. We retrospectively identified >100 cases of iNTS infections in an infectious disease hospital in Southern Vietnam between 2008 and 2013. Clinical records were accessed to evaluate demographic and clinical factors associated with iNTS infection and to identify risk factors associated with death. Multi-locus sequence typing and antimicrobial susceptibility testing was performed on all organisms. Of 102 iNTS patients, 71% were HIV-infected, >90% were adults, 71% were male and 33% reported intravenous drug use. Twenty-six/92 (28%) patients with a known outcome died; HIV infection was significantly associated with death (p = 0.039). S. Enteritidis (Sequence Types (ST)11) (48%, 43/89) and S. Typhimurium (ST19, 34 and 1544) (26%, 23/89) were the most commonly identified serovars; S. Typhimurium was significantly more common in HIV-infected individuals (p = 0.003). Isolates from HIV-infected patients were more likely to exhibit reduced susceptibility against trimethoprim-sulfamethoxazole than HIV-negative patients (p = 0.037). We conclude that iNTS disease is a severe infection in Vietnam with a high mortality rate. As in sub-Saharan Africa, HIV infection was a risk factor for death, with the majority of the burden in this population found in HIV-infected adult men.

Chung The H, Rabaa MA, Pham Thanh D, De Lappe N, Cormican M, Valcanis M, Howden BP, Wangchuk S, Bodhidatta L, Mason CJ et al. 2016. South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study. PLoS Med, 13 (8), pp. e1002055. | Show Abstract | Read more

BACKGROUND: Antimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei. METHODS AND FINDINGS: We performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei. CONCLUSIONS: This study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.

Van Voorhis WC, Adams JH, Adelfio R, Ahyong V, Akabas MH, Alano P, Alday A, Alemán Resto Y, Alsibaee A, Alzualde A et al. 2016. Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond. PLoS Pathog, 12 (7), pp. e1005763. | Show Abstract | Read more

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Pham Thanh D, Thompson CN, Rabaa MA, Sona S, Sopheary S, Kumar V, Moore C, Tran Vu Thieu N, Wijedoru L, Holt KE et al. 2016. The Molecular and Spatial Epidemiology of Typhoid Fever in Rural Cambodia. PLoS Negl Trop Dis, 10 (6), pp. e0004785. | Show Abstract | Read more

Typhoid fever, caused by the bacterium Salmonella Typhi, is an endemic cause of febrile disease in Cambodia. The aim of this study was to better understand the epidemiology of pediatric typhoid fever in Cambodia. We accessed routine blood culture data from Angkor Hospital for Children (AHC) in Siem Reap province between 2007 and 2014, and performed whole genome sequencing (WGS) on the isolated bacteria to characterize the S. Typhi population. The resulting phylogenetic information was combined with conventional epidemiological approaches to investigate the spatiotemporal distribution of S. Typhi and population-level risk factors for reported disease. During the study period, there were 262 cases of typhoid within a 100 km radius of AHC, with a median patient age of 8.2 years (IQR: 5.1-11.5 years). The majority of infections occurred during the rainy season, and commune incidences as high as 11.36/1,000 in children aged <15 years were observed over the study period. A population-based risk factor analysis found that access to water within households and increasing distance from Tonle Sap Lake were protective. Spatial mapping and WGS provided additional resolution for these findings, and confirmed that proximity to the lake was associated with discrete spatiotemporal disease clusters. We confirmed the dominance of MDR H58 S. Typhi in this population, and found substantial evidence of diversification (at least seven sublineages) within this single lineage. We conclude that there is a substantial burden of pediatric typhoid fever in rural communes in Cambodia. Our data provide a platform for additional population-based typhoid fever studies in this location, and suggest that this would be a suitable setting in which to introduce a school-based vaccination programme with Vi conjugate vaccines.

Pham Thanh D, Thanh Tuyen H, Nguyen Thi Nguyen T, Chung The H, Wick RR, Thwaites GE, Baker S, Holt KE. 2016. Inducible colistin resistance via a disrupted plasmid-borne mcr-1 gene in a 2008 Vietnamese Shigella sonnei isolate. J Antimicrob Chemother, 71 (8), pp. 2314-2317. | Show Abstract | Read more

OBJECTIVES: The objective of this study was to assess the presence of mcr-1 in Shigella sonnei isolated in Vietnam. METHODS: WGS data were analysed for the presence of the mcr-1 gene sequence. The association of mcr-1 with a plasmid was assessed by PCR and by conjugation. RESULTS: Through genome sequencing we identified a plasmid-associated inactive form of mcr-1 in a 2008 Vietnamese isolate of Shigella sonnei. The plasmid was conjugated into Escherichia coli and mcr-1 was activated upon exposure to colistin, resulting in highly colistin-resistant transconjugants. CONCLUSIONS: This is the first description of the mcr-1 gene in Shigella, which is atypical given that colistin is not ordinarily used to treat diarrhoea. Our data suggest the mcr-1 gene has been circulating in human-restricted pathogens for some time but likely carries a selective fitness cost.

Trung NV, Carrique-Mas JJ, Nghia NH, Tu LT, Mai HH, Tuyen HT, Campbell J, Nhung NT, Nhung HN, Minh PV et al. 2017. Non-Typhoidal Salmonella Colonization in Chickens and Humans in the Mekong Delta of Vietnam. Zoonoses Public Health, 64 (2), pp. 94-99. | Show Abstract | Read more

Salmonellosis is a public health concern in both the developed and developing countries. Although the majority of human non-typhoidal Salmonella enterica (NTS) cases are the result of foodborne infections or person-to-person transmission, NTS infections may also be acquired by environmental and occupational exposure to animals. While a considerable number of studies have investigated the presence of NTS in farm animals and meat/carcasses, very few studies have investigated the risk of NTS colonization in humans as a result of direct animal exposure. We investigated asymptomatic NTS colonization in 204 backyard chicken farms, 204 farmers and 306 matched individuals not exposed to chicken farming, in southern Vietnam. Pooled chicken faeces, collected using boot or handheld swabs on backyard chicken farms, and rectal swabs from human participants were tested. NTS colonization prevalence was 45.6%, 4.4% and 2.6% for chicken farms, farmers and unexposed individuals, respectively. Our study observed a higher prevalence of NTS colonization among chicken farmers (4.4%) compared with age-, sex- and location- matched rural and urban individuals not exposed to chickens (2.9% and 2.0%). A total of 164 chicken NTS strains and 17 human NTS strains were isolated, and 28 serovars were identified. Salmonella Weltevreden was the predominant serovar in both chickens and humans. NTS isolates showed resistance (20-40%) against tetracycline, chloramphenicol, sulfamethoxazole-trimethoprim and ampicillin. Our study reflects the epidemiology of NTS colonization in chickens and humans in the Mekong delta of Vietnam and emphasizes the need of larger, preferably longitudinal studies to study the transmission dynamics of NTS between and within animal and human host populations.

Nguyen NT, Nguyen HM, Nguyen CV, Nguyen TV, Nguyen MT, Thai HQ, Ho MH, Thwaites G, Ngo HT, Baker S, Carrique-Mas J. 2016. Use of Colistin and Other Critical Antimicrobials on Pig and Chicken Farms in Southern Vietnam and Its Association with Resistance in Commensal Escherichia coli Bacteria. Appl Environ Microbiol, 82 (13), pp. 3727-3735. | Show Abstract | Read more

UNLABELLED: Antimicrobial resistance (AMR) is a global health problem, and emerging semi-intensive farming systems in Southeast Asia are major contributors to the AMR burden. We accessed 12 pig and chicken farms at key stages of production in Tien Giang Province, Vietnam, to measure antimicrobial usage and to investigate the prevalence of AMR to five critical antimicrobials (β-lactams, third-generation cephalosporins, quinolones, aminoglycosides, and polymyxins) and their corresponding molecular mechanisms among 180 Escherichia coli isolates. Overall, 94.7 mg (interquartile range [IQR], 65.3 to 151.1) and 563.6 mg (IQR, 398.9 to 943.6) of antimicrobials was used to produce 1 kg (live weight) of chicken and pig, respectively. A median of 3 (out of 8) critical antimicrobials were used on pig farms. E. coli isolates exhibited a high prevalence of resistance to ampicillin (97.8% and 94.4% for chickens and pigs, respectively), ciprofloxacin (73.3% and 21.1%), gentamicin (42.2% and 35.6%), and colistin (22.2% and 24.4%). The prevalence of a recently discovered colistin resistance gene, mcr-1, was 19 to 22% and had strong agreement with phenotypic colistin resistance. We conducted plasmid conjugation experiments with 37 mcr-1 gene-positive E. coli isolates and successfully observed transfer of the gene in 54.0% of isolates through a plasmid of approximately 63 kb, consistent with one recently identified in China. We found no significant correlation between total use of antimicrobials at the farm level and AMR. These data provide additional insight into the role of mcr-1 in colistin resistance on farms and outline the dynamics of phenotypic and genotypic AMR in semi-intensive farming systems in Vietnam. IMPORTANCE: Our study provides accurate baseline information on levels of antimicrobial use, as well as on the dynamics of phenotypic and genotypic resistance for antimicrobials of critical importance among E. coli over the different stages of production in emerging pig and poultry production systems in Vietnam. E. coli isolates showed a high prevalence of resistance (>20%) to critically important antimicrobials, such as colistin, ciprofloxacin, and gentamicin. The underlying genetic mechanisms identified for colistin (the mcr-1 gene) and quinolone (gyrA gene mutations) are likely to play a major role in AMR to those compounds. Conjugation experiments led to the identification of a 63-kb plasmid, similar to one recently identified in China, as the potential carrier of the mcr-1 gene. These results should encourage greater restrictions of such antimicrobials in Southeast Asian farming systems.

Baker S, Hombach J, Marks F. 2016. What Have We Learned From the Typhoid Fever Surveillance in Africa Program? Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S1-S3. | Show Abstract | Read more

The Typhoid Fever Surveillance in Africa Program (TSAP) was established in 2009 to fill the data void concerning invasive Salmonella disease in sub-Saharan Africa, and to specifically estimate the burden of bloodstream infections caused by the key pathogen, Salmonella enterica serovar Typhi. TSAP has achieved this ambitious target, finding high incidences of typhoid fever in both rural and urban populations in several countries in sub-Saharan Africa. The results of TSAP will undoubtedly dictate the direction of future typhoid fever research in Africa, and at last provides a key piece of the disease burden jigsaw puzzle. With the dawn of new Vi conjugate vaccines against Salmonella Typhi, the next priority for the typhoid community must be providing the required data on these vaccines so they can be licensed and provided to those in high-risk groups and locations across sub-Saharan Africa.

Cruz Espinoza LM, Nichols C, Adu-Sarkodie Y, Al-Emran HM, Baker S, Clemens JD, Dekker DM, Eibach D, Krumkamp R, Boahen K et al. 2016. Variations of Invasive Salmonella Infections by Population Size in Asante Akim North Municipal, Ghana. Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S17-S22. | Show Abstract | Read more

BACKGROUND: The Typhoid Fever Surveillance in Africa Program (TSAP) estimated adjusted incidence rates (IRs) for Salmonella enterica serovar Typhi and invasive nontyphoidal S. enterica serovars (iNTS) of >100 cases per 100 000 person-years of observation (PYO) for children aged <15 years in Asante Akim North Municipal (AAN), Ghana, between March 2010 and May 2012. We analyzed how much these rates differed between rural and urban settings. METHODS: Children recruited at the Agogo Presbyterian Hospital and meeting TSAP inclusion criteria were included in the analysis. Towns with >32 000 inhabitants were considered urban; towns with populations <5200 were considered rural. Adjusted IRs for Salmonella bloodstream infections were estimated for both settings. Setting-specific age-standardized incidence rates for children aged <15 years were derived and used to calculate age-standardized rate ratios (SRRs) to evaluate differences between settings. RESULTS: Eighty-eight percent (2651/3000) of recruited patients met inclusion criteria and were analyzed. IRs of Salmonella bloodstream infections in children <15 years old were >100 per 100 000 PYO in both settings. Among rural children, the Salmonella Typhi and iNTS rates were 2 times (SRR, 2.2; 95% confidence interval [CI], 1.3-3.5) and almost 3 times (SRR, 2.8; 95% CI, 1.9-4.3) higher, respectively, than rates in urban children. CONCLUSIONS: IRs of Salmonella bloodstream infections in children <15 years old in AAN, Ghana, differed by setting, with 2 to nearly 3 times higher rates in the less populated setting. Variations in the distribution of the disease should be considered to implement future studies and intervention strategies.

Al-Emran HM, Eibach D, Krumkamp R, Ali M, Baker S, Biggs HM, Bjerregaard-Andersen M, Breiman RF, Clemens JD, Crump JA et al. 2016. A Multicountry Molecular Analysis of Salmonella enterica Serovar Typhi With Reduced Susceptibility to Ciprofloxacin in Sub-Saharan Africa. Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S42-S46. | Show Abstract | Read more

BACKGROUND: Salmonella enterica serovar Typhi is a predominant cause of bloodstream infections in sub-Saharan Africa (SSA). Increasing numbers of S. Typhi with resistance to ciprofloxacin have been reported from different parts of the world. However, data from SSA are limited. In this study, we aimed to measure the ciprofloxacin susceptibility of S. Typhi isolated from patients with febrile illness in SSA. METHODS: Febrile patients from 9 sites within 6 countries in SSA with a body temperature of ≥38.0°C were enrolled in this study. Blood samples were obtained for bacterial culture, and Salmonella isolates were identified biochemically and confirmed by multiplex polymerase chain reaction (PCR). Antimicrobial susceptibility of all Salmonella isolates was performed by disk diffusion test, and minimum inhibitory concentrations (MICs) against ciprofloxacin were measured by Etest. All Salmonella isolates with reduced susceptibility to ciprofloxacin (MIC > 0.06 µg/mL) were screened for mutations in quinolone resistance-determining regions in target genes, and the presence of plasmid-mediated quinolone resistance (PMQR) genes was assessed by PCR. RESULTS: A total of 8161 blood cultures were performed, and 100 (1.2%) S. Typhi, 2 (<0.1%) Salmonella enterica serovar Paratyphi A, and 27 (0.3%) nontyphoid Salmonella (NTS) were isolated. Multidrug-resistant S. Typhi were isolated in Kenya (79% [n = 38]) and Tanzania (89% [n = 8]) only. Reduced ciprofloxacin-susceptible (22% [n = 11]) S. Typhi were isolated only in Kenya. Among those 11 isolates, all had a Glu133Gly mutation in the gyrA gene combined with either a gyrA (Ser83Phe) or gyrB mutation (Ser464Phe). One Salmonella Paratyphi A isolate with reduced susceptibility to ciprofloxacin was found in Senegal, with 1 mutation in gyrA (Ser83Phe) and a second mutation in parC (Ser57Phe). Mutations in the parE gene and PMQR genes were not detected in any isolate. CONCLUSIONS: Salmonella Typhi with reduced susceptibility to ciprofloxacin was not distributed homogenously throughout SSA. Its prevalence was very high in Kenya, and was not observed in other study countries. Continuous monitoring of antimicrobial susceptibility is required to follow the potential spread of antimicrobial-resistant isolates throughout SSA.

Im J, Nichols C, Bjerregaard-Andersen M, Sow AG, Løfberg S, Tall A, Pak GD, Aaby P, Baker S, Clemens JD et al. 2016. Prevalence of Salmonella Excretion in Stool: A Community Survey in 2 Sites, Guinea-Bissau and Senegal. Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S50-S55. | Show Abstract | Read more

BACKGROUND: Chronic and convalescent carriers play an important role in the transmission and endemicity of many communicable diseases. A high incidence of Salmonella enterica serovar Typhi and invasive nontyphoidal Salmonella (NTS) infection has been reported in parts of sub-Saharan Africa, yet the prevalence of Salmonella excretion in the general population is unknown. METHODS: Stool specimens were collected from a random sample of households in 2 populations in West Africa: Bissau, Guinea-Bissau, and Dakar, Senegal. Stool was cultured to detect presence of Salmonella, and antimicrobial susceptibility testing was performed on the isolated organisms. RESULTS: Stool was cultured from 1077 and 1359 individuals from Guinea-Bissau and Senegal, respectively. Salmonella Typhi was not isolated from stool samples at either site. Prevalence of NTS in stool samples was 24.1 (95% confidence interval [CI], 16.5-35.1; n = 26/1077) per 1000 population in Guinea-Bissau and 10.3 (95% CI, 6.1-17.2; n = 14/1359) per 1000 population in Senegal. CONCLUSIONS: Evidence of NTS excretion in stool in both study populations indicates a possible NTS transmission route in these settings.

Panzner U, Pak GD, Aaby P, Adu-Sarkodie Y, Ali M, Aseffa A, Baker S, Bjerregaard-Andersen M, Crump JA, Deerin J et al. 2016. Utilization of Healthcare in the Typhoid Fever Surveillance in Africa Program. Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S56-S68. | Show Abstract | Read more

BACKGROUND: Assessing healthcare utilization is important to identify weaknesses of healthcare systems, to outline action points for preventive measures and interventions, and to more accurately estimate the disease burden in a population. METHODS: A healthcare utilization survey was developed for the Typhoid Fever Surveillance in Africa Program (TSAP) to adjust incidences of salmonellosis determined through passive, healthcare facility-based surveillance. This cross-sectional survey was conducted at 11 sites in 9 sub-Saharan African countries. Demographic data and healthcare-seeking behavior were assessed at selected households. Overall and age-stratified percentages of each study population that sought healthcare at a TSAP healthcare facility and elsewhere were determined. RESULTS: Overall, 88% (1007/1145) and 81% (1811/2238) of the population in Polesgo and Nioko 2, Burkina Faso, respectively, and 63% (1636/2590) in Butajira, Ethiopia, sought healthcare for fever at any TSAP healthcare facility. A far smaller proportion-namely, 20%-45% of the population in Bissau, Guinea-Bissau (1743/3885), Pikine, Senegal (1473/4659), Wad-Medani, Sudan (861/3169), and Pietermaritzburg, South Africa (667/2819); 18% (483/2622) and 9% (197/2293) in Imerintsiatosika and Isotry, Madagascar, respectively; and 4% (127/3089) in Moshi, Tanzania-sought healthcare at a TSAP healthcare facility. Patients with fever preferred to visit pharmacies in Imerintsiatosika and Isotry, and favored self-management of fever in Moshi. Age-dependent differences in healthcare utilization were also observed within and across sites. CONCLUSIONS: Healthcare utilization for fever varied greatly across sites, and revealed that not all studied populations were under optimal surveillance. This demonstrates the importance of assessing healthcare utilization. Survey data were pivotal for the adjustment of the program's estimates of salmonellosis and other conditions associated with fever.

Pach A, Warren M, Chang I, Im J, Nichols C, Meyer CG, Pak GD, Panzner U, Park SE, von Kalckreuth V et al. 2016. A Qualitative Study Investigating Experiences, Perceptions, and Healthcare System Performance in Relation to the Surveillance of Typhoid Fever in Madagascar. Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S69-S75. | Show Abstract | Read more

BACKGROUND: The burden of typhoid fever (TF) in sub-Saharan Africa is largely unknown but is increasingly thought to be high, given that water and sanitary conditions remain unimproved in many countries. To address this gap in information, the Typhoid Fever Surveillance in Africa Program (TSAP) founded a surveillance system for TF in 10 African countries. This study was a component of the TSAP surveillance project in Madagascar. METHODS: The study entailed a qualitative assessment of patients' experiences and perceptions of services for febrile symptoms at the studies' rural and urban sentinel public health clinics. The study examined influences on the use of these facilities, alternative sources of care, and providers' descriptions of medical consultations and challenges in providing services. Data were collected through semistructured and open-ended individual interviews and a focus group with patients, caregivers, and medical personnel. RESULTS: Thirty-three patients and 12 healthcare providers participated in the data collection across the 2 healthcare facilities. The quality of services, cost, and travel distance were key factors that enabled access to and use of these clinics. Divergent healthcare-seeking patterns were related to variability in the care utilized, socioeconomic status, and potential distance from the facilities : These factors influenced delivery of care, patient access, and the health facilities' capacity to identify cases of febrile illness such as TF. CONCLUSIONS: This approach provided an in-depth investigation and understanding of healthcare-seeking behavior at the study facilities, and factors that facilitated or acted as barriers to their use. Our findings demonstrate the relevance of these public health clinics as sites for the surveillance of TF in their role as central healthcare sources for families and communities within these rural and urban areas of Madagascar.

Marks F, Rabehanta N, Baker S, Panzner U, Park SE, Fobil JN, Meyer CG, Rakotozandrindrainy R. 2016. A Way Forward for Healthcare in Madagascar? Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S76-S79. | Show Abstract | Read more

A healthcare utilization survey was conducted as a component of the Typhoid Fever Surveillance in Africa Program (TSAP). The findings of this survey in Madagascar contrasted with those in other sites of the program; namely, only 30% of the population sought healthcare at the government-provided healthcare facilities for fever. These findings promoted us to determine the drivers and barriers in accessing and utilizing healthcare in Madagascar. Here we review the results of the TSAP healthcare utilization initiative and place them in the context of the current organization of the Madagascan healthcare system. Our work highlights the demands of the population for access to appropriate healthcare and the need for novel solutions that can quickly provide an affordable and sustainable basic healthcare infrastructure until a government-funded scheme is in place.

von Kalckreuth V, Konings F, Aaby P, Adu-Sarkodie Y, Ali M, Aseffa A, Baker S, Breiman RF, Bjerregaard-Andersen M, Clemens JD et al. 2016. The Typhoid Fever Surveillance in Africa Program (TSAP): Clinical, Diagnostic, and Epidemiological Methodologies. Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S9-S16. | Show Abstract | Read more

BACKGROUND: New immunization programs are dependent on data from surveillance networks and disease burden estimates to prioritize target areas and risk groups. Data regarding invasive Salmonella disease in sub-Saharan Africa are currently limited, thus hindering the implementation of preventive measures. The Typhoid Fever Surveillance in Africa Program (TSAP) was established by the International Vaccine Institute to obtain comparable incidence data on typhoid fever and invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa through standardized surveillance in multiple countries. METHODS: Standardized procedures were developed and deployed across sites for study site selection, patient enrolment, laboratory procedures, quality control and quality assurance, assessment of healthcare utilization and incidence calculations. RESULTS: Passive surveillance for bloodstream infections among febrile patients was initiated at thirteen sentinel sites in ten countries (Burkina Faso, Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar, Senegal, South Africa, Sudan, and Tanzania). Each TSAP site conducted case detection using these standardized methods to isolate and identify aerobic bacteria from the bloodstream of febrile patients. Healthcare utilization surveys were conducted to adjust population denominators in incidence calculations for differing healthcare utilization patterns and improve comparability of incidence rates across sites. CONCLUSIONS: By providing standardized data on the incidence of typhoid fever and iNTS disease in sub-Saharan Africa, TSAP will provide vital input for targeted typhoid fever prevention programs.

Park SE, Pak GD, Aaby P, Adu-Sarkodie Y, Ali M, Aseffa A, Biggs HM, Bjerregaard-Andersen M, Breiman RF, Crump JA et al. 2016. The Relationship Between Invasive Nontyphoidal Salmonella Disease, Other Bacterial Bloodstream Infections, and Malaria in Sub-Saharan Africa. Clin Infect Dis, 62 Suppl 1 (suppl 1), pp. S23-S31. | Show Abstract | Read more

BACKGROUND: Country-specific studies in Africa have indicated that Plasmodium falciparum is associated with invasive nontyphoidal Salmonella (iNTS) disease. We conducted a multicenter study in 13 sites in Burkina Faso, Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar, Senegal, South Africa, Sudan, and Tanzania to investigate the relationship between the occurrence of iNTS disease, other systemic bacterial infections, and malaria. METHODS: Febrile patients received a blood culture and a malaria test. Isolated bacteria underwent antimicrobial susceptibility testing, and the association between iNTS disease and malaria was assessed. RESULTS: A positive correlation between frequency proportions of malaria and iNTS was observed (P = .01; r = 0.70). Areas with higher burden of malaria exhibited higher odds of iNTS disease compared to other bacterial infections (odds ratio [OR], 4.89; 95% CI, 1.61-14.90; P = .005) than areas with lower malaria burden. Malaria parasite positivity was associated with iNTS disease (OR, 2.44; P = .031) and gram-positive bacteremias, particularly Staphylococcus aureus, exhibited a high proportion of coinfection with Plasmodium malaria. Salmonella Typhimurium and Salmonella Enteritidis were the predominant NTS serovars (53/73; 73%). Both moderate (OR, 6.05; P = .0001) and severe (OR, 14.62; P < .0001) anemia were associated with iNTS disease. CONCLUSIONS: A positive correlation between iNTS disease and malaria endemicity, and the association between Plasmodium parasite positivity and iNTS disease across sub-Saharan Africa, indicates the necessity to consider iNTS as a major cause of febrile illness in malaria-holoendemic areas. Prevention of iNTS disease through iNTS vaccines for areas of high malaria endemicity, targeting high-risk groups for Plasmodium parasitic infection, should be considered.

Schultz MB, Pham Thanh D, Tran Do Hoan N, Wick RR, Ingle DJ, Hawkey J, Edwards DJ, Kenyon JJ, Phu Huong Lan N, Campbell JI et al. 2016. Repeated local emergence of carbapenem-resistant Acinetobacter baumannii in a single hospital ward. Microb Genom, 2 (3), pp. e000050. | Show Abstract | Read more

We recently reported a dramatic increase in the prevalence of carbapenem-resistant Acinetobacter baumannii infections in the intensive care unit (ICU) of a Vietnamese hospital. This upsurge was associated with a specific oxa23-positive clone that was identified by multilocus VNTR analysis. Here, we used whole-genome sequence analysis to dissect the emergence of carbapenem-resistant A. baumannii causing ventilator-associated pneumonia (VAP) in the ICU during 2009-2012. To provide historical context and distinguish microevolution from strain introduction, we compared these genomes with those of A. baumannii asymptomatic carriage and VAP isolates from this same ICU collected during 2003-2007. We identified diverse lineages co-circulating over many years. Carbapenem resistance was associated with the presence of oxa23, oxa40, oxa58 and ndm1 genes in multiple lineages. The majority of resistant isolates were oxa23-positive global clone GC2; fine-scale phylogenomic analysis revealed five distinct GC2 sublineages within the ICU that had evolved locally via independent chromosomal insertions of oxa23 transposons. The increase in infections caused by carbapenem-resistant A. baumannii was associated with transposon-mediated transmission of a carbapenemase gene, rather than clonal expansion or spread of a carbapenemase-harbouring plasmid. Additionally, we found evidence of homologous recombination creating diversity within the local GC2 population, including several events resulting in replacement of the capsule locus. We identified likely donors of the imported capsule locus sequences amongst the A. baumannii isolated on the same ward, suggesting that diversification was largely facilitated via reassortment and sharing of genetic material within the localized A. baumannii population.

The HC, Thanh DP, Holt KE, Thomson NR, Baker S. 2016. The genomic signatures of Shigella evolution, adaptation and geographical spread. Nat Rev Microbiol, 14 (4), pp. 235-250. | Show Abstract | Read more

Shigella spp. are some of the key pathogens responsible for the global burden of diarrhoeal disease. These facultative intracellular bacteria belong to the family Enterobacteriaceae, together with other intestinal pathogens, such as Escherichia coli and Salmonella spp. The genus Shigella comprises four different species, each consisting of several serogroups, all of which show phenotypic similarity, including invasive pathogenicity. DNA sequencing suggests that this similarity results from the convergent evolution of different Shigella spp. founders. Here, we review the evolutionary relationships between Shigella spp. and E . coli, and we highlight how the genomic plasticity of these bacteria and their acquisition of a distinctive virulence plasmid have enabled the development of such highly specialized pathogens. Furthermore, we discuss the insights that genotyping and whole-genome sequencing have provided into the phylogenetics and intercontinental spread of Shigella spp.

Duong VT, Phat VV, Tuyen HT, Dung TT, Trung PD, Minh PV, Tu LETP, Campbell JI, Le Phuc H, Ha TT et al. 2016. Evaluation of Luminex xTAG Gastrointestinal Pathogen Panel Assay for Detection of Multiple Diarrheal Pathogens in Fecal Samples in Vietnam. J Clin Microbiol, 54 (4), pp. 1094-1100. | Show Abstract | Read more

Diarrheal disease is a complex syndrome that remains a leading cause of global childhood morbidity and mortality. The diagnosis of enteric pathogens in a timely and precise manner is important for making treatment decisions and informing public health policy, but accurate diagnosis is a major challenge in industrializing countries. Multiplex molecular diagnostic techniques may represent a significant improvement over classical approaches. We evaluated the Luminex xTAG gastrointestinal pathogen panel (GPP) assay for the detection of common enteric bacterial and viral pathogens in Vietnam. Microbiological culture and real-time PCR were used as gold standards. The tests were performed on 479 stool samples collected from people admitted to the hospital for diarrheal disease throughout Vietnam. Sensitivity and specificity were calculated for the xTAG GPP for the seven principal diarrheal etiologies. The sensitivity and specificity for the xTAG GPP were >88% for Shigellaspp.,Campylobacterspp., rotavirus, norovirus genotype 1/2 (GI/GII), and adenovirus compared to those of microbiological culture and/or real-time PCR. However, the specificity was low (∼60%) for Salmonella species. Additionally, a number of important pathogens that are not identified in routine hospital procedures in this setting, such as Cryptosporidiumspp. and Clostridium difficile, were detected with the GPP. The use of the Luminex xTAG GPP for the detection of enteric pathogens in settings, like Vietnam, would dramatically improve the diagnostic accuracy and capacity of hospital laboratories, allowing for timely and appropriate therapy decisions and a wider understanding of the epidemiology of pathogens associated with severe diarrheal disease in low-resource settings.

Van Vinh Chau N, Buu Chau L, Desquesnes M, Herder S, Phu Huong Lan N, Campbell JI, Van Cuong N, Yimming B, Chalermwong P, Jittapalapong S et al. 2016. A Clinical and Epidemiological Investigation of the First Reported Human Infection With the Zoonotic Parasite Trypanosoma evansi in Southeast Asia. Clin Infect Dis, 62 (8), pp. 1002-1008. | Show Abstract | Read more

BACKGROUND: Trypanosomais a genus of unicellular parasitic flagellate protozoa.Trypanosoma bruceispecies and Trypanosoma cruziare the major agents of human trypanosomiasis; other Trypanosomaspecies can cause human disease, but are rare. In March 2015, a 38-year-old woman presented to a healthcare facility in southern Vietnam with fever, headache, and arthralgia. Microscopic examination of blood revealed infection with Trypanosoma METHODS: Microscopic observation, polymerase chain reaction (PCR) amplification of blood samples, and serological testing were performed to identify the infecting species. The patient's blood was screened for the trypanocidal protein apolipoprotein L1 (APOL1), and a field investigation was performed to identify the zoonotic source. RESULTS: PCR amplification and serological testing identified the infecting species as Trypanosoma evansi.Despite relapsing 6 weeks after completing amphotericin B therapy, the patient made a complete recovery after 5 weeks of suramin. The patient was found to have 2 wild-type APOL1 alleles and a normal serum APOL1 concentration. After responsive animal sampling in the presumed location of exposure, cattle and/or buffalo were determined to be the most likely source of the infection, with 14 of 30 (47%) animal blood samples testing PCR positive forT. evansi. CONCLUSIONS: We report the first laboratory-confirmed case ofT. evansiin a previously healthy individual without APOL1 deficiency, potentially contracted via a wound while butchering raw beef, and successfully treated with suramin. A linked epidemiological investigation revealed widespread and previously unidentified burden ofT. evansiin local cattle, highlighting the need for surveillance of this infection in animals and the possibility of further human cases.

Makendi C, Page AJ, Wren BW, Le Thi Phuong T, Clare S, Hale C, Goulding D, Klemm EJ, Pickard D, Okoro C et al. 2016. A Phylogenetic and Phenotypic Analysis of Salmonella enterica Serovar Weltevreden, an Emerging Agent of Diarrheal Disease in Tropical Regions. PLoS Negl Trop Dis, 10 (2), pp. e0004446. | Show Abstract | Read more

Salmonella enterica serovar Weltevreden (S. Weltevreden) is an emerging cause of diarrheal and invasive disease in humans residing in tropical regions. Despite the regional and international emergence of this Salmonella serovar, relatively little is known about its genetic diversity, genomics or virulence potential in model systems. Here we used whole genome sequencing and bioinformatics analyses to define the phylogenetic structure of a diverse global selection of S. Weltevreden. Phylogenetic analysis of more than 100 isolates demonstrated that the population of S. Weltevreden can be segregated into two main phylogenetic clusters, one associated predominantly with continental Southeast Asia and the other more internationally dispersed. Subcluster analysis suggested the local evolution of S. Weltevreden within specific geographical regions. Four of the isolates were sequenced using long read sequencing to produce high quality reference genomes. Phenotypic analysis in Hep-2 cells and in a murine infection model indicated that S. Weltevreden were significantly attenuated in these models compared to the classical S. Typhimurium reference strain SL1344. Our work outlines novel insights into this important emerging pathogen and provides a baseline understanding for future research studies.

Arjyal A, Basnyat B, Nhan HT, Koirala S, Giri A, Joshi N, Shakya M, Pathak KR, Mahat SP, Prajapati SP et al. 2016. Gatifloxacin versus ceftriaxone for uncomplicated enteric fever in Nepal: an open-label, two-centre, randomised controlled trial. Lancet Infect Dis, 16 (5), pp. 535-545. | Show Abstract | Read more

BACKGROUND: Because treatment with third-generation cephalosporins is associated with slow clinical improvement and high relapse burden for enteric fever, whereas the fluoroquinolone gatifloxacin is associated with rapid fever clearance and low relapse burden, we postulated that gatifloxacin would be superior to the cephalosporin ceftriaxone in treating enteric fever. METHODS: We did an open-label, randomised, controlled, superiority trial at two hospitals in the Kathmandu valley, Nepal. Eligible participants were children (aged 2-13 years) and adult (aged 14-45 years) with criteria for suspected enteric fever (body temperature ≥38·0°C for ≥4 days without a focus of infection). We randomly assigned eligible patients (1:1) without stratification to 7 days of either oral gatifloxacin (10 mg/kg per day) or intravenous ceftriaxone (60 mg/kg up to 2 g per day for patients aged 2-13 years, or 2 g per day for patients aged ≥14 years). The randomisation list was computer-generated using blocks of four and six. The primary outcome was a composite of treatment failure, defined as the occurrence of at least one of the following: fever clearance time of more than 7 days after treatment initiation; the need for rescue treatment on day 8; microbiological failure (ie, blood cultures positive for Salmonella enterica serotype Typhi, or Paratyphi A, B, or C) on day 8; or relapse or disease-related complications within 28 days of treatment initiation. We did the analyses in the modified intention-to-treat population, and subpopulations with either confirmed blood-culture positivity, or blood-culture negativity. The trial was powered to detect an increase of 20% in the risk of failure. This trial was registered at, number NCT01421693, and is now closed. FINDINGS: Between Sept 18, 2011, and July 14, 2014, we screened 725 patients for eligibility. On July 14, 2014, the trial was stopped early by the data safety and monitoring board because S Typhi strains with high-level resistance to ciprofloxacin and gatifloxacin had emerged. At this point, 239 were in the modified intention-to-treat population (120 assigned to gatifloxacin, 119 to ceftriaxone). 18 (15%) patients who received gatifloxacin had treatment failure, compared with 19 (16%) who received ceftriaxone (hazard ratio [HR] 1·04 [95% CI 0·55-1·98]; p=0·91). In the culture-confirmed population, 16 (26%) of 62 patients who received gatifloxacin failed treatment, compared with four (7%) of 54 who received ceftriaxone (HR 0·24 [95% CI 0·08-0·73]; p=0·01). Treatment failure was associated with the emergence of S Typhi exhibiting resistance against fluoroquinolones, requiring the trial to be stopped. By contrast, in patients with a negative blood culture, only two (3%) of 58 who received gatifloxacin failed treatment versus 15 (23%) of 65 who received ceftriaxone (HR 7·50 [95% CI 1·71-32·80]; p=0·01). A similar number of non-serious adverse events occurred in each treatment group, and no serious events were reported. INTERPRETATION: Our results suggest that fluoroquinolones should no longer be used for treatment of enteric fever in Nepal. Additionally, under our study conditions, ceftriaxone was suboptimum in a high proportion of patients with culture-negative enteric fever. Since antimicrobials, specifically fluoroquinolones, are one of the only routinely used control measures for enteric fever, the assessment of novel diagnostics, new treatment options, and use of existing vaccines and development of next-generation vaccines are now a high priority. FUNDING: Wellcome Trust and Li Ka Shing Foundation.

Oude Munnink BB, Phan MVT, van der Hoek L, Kellam P, Cotten M. 2016. Genome Sequences of a Novel Vietnamese Bat Bunyavirus Genome Announcements, 4 (6), pp. e01366-16-e01366-16. | Read more

Lu L, Van Dung N, Bryant JE, Carrique-Mas J, Van Cuong N, Anh PH, Rabaa MA, Baker S, Simmonds P, Woolhouse ME. 2016. Evolution and phylogeographic dissemination of endemic porcine picornaviruses in Vietnam. Virus Evol, 2 (1), pp. vew001. | Show Abstract | Read more

Members of the Picornaviridae are important and often zoonotic viruses responsible for a variety of human and animal diseases. However, the evolution and spatial dissemination of different picornaviruses circulating in domestic animals are not well studied. We examined the rate of evolution and time of origin of porcine enterovirus G (EV-G) and porcine kobuvirus species C lineages (PKV-C) circulating in pig farms in Vietnam and from other countries. We further explored the spatiotemporal spread of EV-G and PKV-C in Southwest Vietnam using phylogeographic models. Multiple types of EV-G are co-circulating in Vietnam. The two dominant EV-G types among isolates from Vietnam (G1 and G6) showed strong phylogenetic clustering. Three clades of PKV-C (PKV-C1-3) represent more recent introductions into Vietnam; PKV-C2 is closely related to PKV-C from Southwest China, indicating possible cross-border dissemination. In addition, high virus lineage migration rates were estimated within four districts in Dong Thap province in Vietnam for both EV-G types (G1, G6) and all PKV-C (C1-3) clades. We found that Chau Thanh district is a primary source of both EV-G and PKV-C clades, consistent with extensive pig trading in and out of the district. Understanding the evolution and spatial dissemination of endemic picornaviruses in pigs may inform future strategies for the surveillance and control of picornaviruses.

Hassan KA, Cain AK, Huang T, Liu Q, Elbourne LD, Boinett CJ, Brzoska AJ, Li L, Ostrowski M, Nhu NT et al. 2016. Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii. MBio, 7 (5), pp. e01200-16-e01200-16. | Show Abstract | Read more

UNLABELLED: Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii Furthermore, the method identified a new transcriptional regulator that controls expression of amvA In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii IMPORTANCE: Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various selective conditions. We applied fluorescence-activated cell sorting (FACS) to physically enrich for phenotypes of interest within a mutant population prior to TraDIS. To our knowledge, this is the first time that a physical selection method has been applied in parallel with TraDIS rather than a fitness-induced selection. The results demonstrate the feasibility of this combined approach to generate significant results and highlight the major multidrug efflux pumps encoded in an important pathogen. This FACS-based approach, TraDISort, could have a range of future applications, including the characterization of efflux pump inhibitors, the identification of regulatory factors controlling gene or protein expression using fluorescent reporters, and the identification of genes involved in cell replication, morphology, and aggregation.

Thi Khanh Nhu N, Riordan DW, Do Hoang Nhu T, Thanh DP, Thwaites G, Huong Lan NP, Wren BW, Baker S, Stabler RA. 2016. The induction and identification of novel Colistin resistance mutations in Acinetobacter baumannii and their implications. Sci Rep, 6 (1), pp. 28291. | Show Abstract | Read more

Acinetobacter baumannii is a significant cause of opportunistic hospital acquired infection and has been identified as an important emerging infection due to its high levels of antimicrobial resistance. Multidrug resistant A. baumannii has risen rapidly in Vietnam, where colistin is becoming the drug of last resort for many infections. In this study we generated spontaneous colistin resistant progeny (up to >256 μg/μl) from four colistin susceptible Vietnamese isolates and one susceptible reference strain (MIC <1.5 μg/μl). Whole genome sequencing was used to identify single nucleotide mutations that could be attributed to the reduced colistin susceptibility. We identified six lpxACD and three pmrB mutations, the majority of which were novel. In addition, we identified further mutations in six A. baumannii genes (vacJ, pldA, ttg2C, pheS and conserved hypothetical protein) that we hypothesise have a role in reduced colistin susceptibility. This study has identified additional mutations that may be associated with colistin resistance through novel resistance mechanisms. Our work further demonstrates how rapidly A. baumannii can generate resistance to a last resort antimicrobial and highlights the need for improved surveillance to identified A. baumannii with an extensive drug resistance profile.

Leung JM, Hong CT, Trung NH, Thi HN, Minh CN, Thi TV, Hong DT, Man DN, Knowles SC, Wolbers M et al. 2016. The impact of albendazole treatment on the incidence of viral- and bacterial-induced diarrhea in school children in southern Vietnam: study protocol for a randomized controlled trial. Trials, 17 (1), pp. 279. | Show Abstract | Read more

BACKGROUND: Anthelmintics are one of the more commonly available classes of drugs to treat infections by parasitic helminths (especially nematodes) in the human intestinal tract. As a result of their cost-effectiveness, mass school-based deworming programs are becoming routine practice in developing countries. However, experimental and clinical evidence suggests that anthelmintic treatments may increase susceptibility to other gastrointestinal infections caused by bacteria, viruses, or protozoa. Hypothesizing that anthelmintics may increase diarrheal infections in treated children, we aim to evaluate the impact of anthelmintics on the incidence of diarrheal disease caused by viral and bacterial pathogens in school children in southern Vietnam. METHODS/DESIGN: This is a randomized, double-blinded, placebo-controlled trial to investigate the effects of albendazole treatment versus placebo on the incidence of viral- and bacterial-induced diarrhea in 350 helminth-infected and 350 helminth-uninfected Vietnamese school children aged 6-15 years. Four hundred milligrams of albendazole, or placebo treatment will be administered once every 3 months for 12 months. At the end of 12 months, all participants will receive albendazole treatment. The primary endpoint of this study is the incidence of diarrheal disease assessed by 12 months of weekly active and passive case surveillance. Secondary endpoints include the prevalence and intensities of helminth, viral, and bacterial infections, alterations in host immunity and the gut microbiota with helminth and pathogen clearance, changes in mean z scores of body weight indices over time, and the number and severity of adverse events. DISCUSSION: In order to reduce helminth burdens, anthelmintics are being routinely administered to children in developing countries. However, the effects of anthelmintic treatment on susceptibility to other diseases, including diarrheal pathogens, remain unknown. It is important to monitor for unintended consequences of drug treatments in co-infected populations. In this trial, we will examine how anthelmintic treatment impacts host susceptibility to diarrheal infections, with the aim of informing deworming programs of any indirect effects of mass anthelmintic administrations on co-infecting enteric pathogens. TRIAL REGISTRATION: NCT02597556 . Registered on 3 November 2015.

Pham Thanh D, Karkey A, Dongol S, Ho Thi N, Thompson CN, Rabaa MA, Arjyal A, Holt KE, Wong V, Tran Vu Thieu N et al. 2016. A novel ciprofloxacin-resistant subclade of H58 Salmonella Typhi is associated with fluoroquinolone treatment failure. Elife, 5 (MARCH2016), pp. e14003. | Show Abstract | Read more

The interplay between bacterial antimicrobial susceptibility, phylogenetics and patient outcome is poorly understood. During a typhoid clinical treatment trial in Nepal, we observed several treatment failures and isolated highly fluoroquinolone-resistant Salmonella Typhi (S. Typhi). Seventy-eight S. Typhi isolates were genome sequenced and clinical observations, treatment failures and fever clearance times (FCTs) were stratified by lineage. Most fluoroquinolone-resistant S. Typhi belonged to a specific H58 subclade. Treatment failure with S. Typhi-H58 was significantly less frequent with ceftriaxone (3/31; 9.7%) than gatifloxacin (15/34; 44.1%)(Hazard Ratio 0.19, p=0.002). Further, for gatifloxacin-treated patients, those infected with fluoroquinolone-resistant organisms had significantly higher median FCTs (8.2 days) than those infected with susceptible (2.96) or intermediately resistant organisms (4.01)(pS. Typhi clade internationally, but there are no data regarding disease outcome with this organism. We report an emergent new subclade of S. Typhi-H58 that is associated with fluoroquinolone treatment failure.

Karkey A, Jombart T, Walker AW, Thompson CN, Torres A, Dongol S, Tran Vu Thieu N, Pham Thanh D, Tran Thi Ngoc D, Voong Vinh P et al. 2016. The Ecological Dynamics of Fecal Contamination and Salmonella Typhi and Salmonella Paratyphi A in Municipal Kathmandu Drinking Water. PLoS Negl Trop Dis, 10 (1), pp. e0004346. | Show Abstract | Read more

One of the UN sustainable development goals is to achieve universal access to safe and affordable drinking water by 2030. It is locations like Kathmandu, Nepal, a densely populated city in South Asia with endemic typhoid fever, where this goal is most pertinent. Aiming to understand the public health implications of water quality in Kathmandu we subjected weekly water samples from 10 sources for one year to a range of chemical and bacteriological analyses. We additionally aimed to detect the etiological agents of typhoid fever and longitudinally assess microbial diversity by 16S rRNA gene surveying. We found that the majority of water sources exhibited chemical and bacterial contamination exceeding WHO guidelines. Further analysis of the chemical and bacterial data indicated site-specific pollution, symptomatic of highly localized fecal contamination. Rainfall was found to be a key driver of this fecal contamination, correlating with nitrates and evidence of S. Typhi and S. Paratyphi A, for which DNA was detectable in 333 (77%) and 303 (70%) of 432 water samples, respectively. 16S rRNA gene surveying outlined a spectrum of fecal bacteria in the contaminated water, forming complex communities again displaying location-specific temporal signatures. Our data signify that the municipal water in Kathmandu is a predominant vehicle for the transmission of S. Typhi and S. Paratyphi A. This study represents the first extensive spatiotemporal investigation of water pollution in an endemic typhoid fever setting and implicates highly localized human waste as the major contributor to poor water quality in the Kathmandu Valley.

Thompson CN, Le TP, Anders KL, Nguyen TH, Lu LV, Nguyen VV, Vu TD, Nguyen NM, Tran TH, Ha TT et al. 2016. The transfer and decay of maternal antibody against Shigella sonnei in a longitudinal cohort of Vietnamese infants. Vaccine, 34 (6), pp. 783-790. | Show Abstract | Read more

BACKGROUND: Shigella sonnei is an emergent and major diarrheal pathogen for which there is currently no vaccine. We aimed to quantify duration of maternal antibody against S. sonnei and investigate transplacental IgG transfer in a birth cohort in southern Vietnam. METHODS AND RESULTS: Over 500-paired maternal/infant plasma samples were evaluated for presence of anti-S. sonnei-O IgG and IgM. Longitudinal plasma samples allowed for the estimation of the median half-life of maternal anti-S. sonnei-O IgG, which was 43 days (95% confidence interval: 41-45 days). Additionally, half of infants lacked a detectable titer by 19 weeks of age. Lower cord titers were associated with greater increases in S. sonnei IgG over the first year of life, and the incidence of S. sonnei seroconversion was estimated to be 4/100 infant years. Maternal IgG titer, the ratio of antibody transfer, the season of birth and gestational age were significantly associated with cord titer. CONCLUSIONS: Maternal anti-S. sonnei-O IgG is efficiently transferred across the placenta and anti-S. sonnei-O maternal IgG declines rapidly after birth and is undetectable after 5 months in the majority of children. Preterm neonates and children born to mothers with low IgG titers have lower cord titers and therefore may be at greater risk of seroconversion in infancy.

Thompson CN, Thieu NT, Vinh PV, Duc AN, Wolbers M, Vinh H, Campbell JI, Ngoc DT, Hoang NV, Thanh TH et al. 2016. Clinical implications of reduced susceptibility to fluoroquinolones in paediatric Shigella sonnei and Shigella flexneri infections. J Antimicrob Chemother, 71 (3), pp. 807-815. | Show Abstract | Read more

OBJECTIVES: We aimed to quantify the impact of fluoroquinolone resistance on the clinical outcome of paediatric shigellosis patients treated with fluoroquinolones in southern Vietnam. Such information is important to inform therapeutic management for infections caused by this increasingly drug-resistant pathogen, responsible for high morbidity and mortality in young children globally. METHODS: Clinical information and bacterial isolates were derived from a randomized controlled trial comparing gatifloxacin with ciprofloxacin for the treatment of paediatric shigellosis. Time-kill experiments were performed to evaluate the impact of MIC on the in vitro growth of Shigella and Cox regression modelling was used to compare clinical outcome between treatments and Shigella species. RESULTS: Shigella flexneri patients treated with gatifloxacin had significantly worse outcomes than those treated with ciprofloxacin. However, the MICs of fluoroquinolones were not significantly associated with poorer outcome. The presence of S83L and A87T mutations in the gyrA gene significantly increased MICs of fluoroquinolones. Finally, elevated MICs and the presence of the qnrS gene allowed Shigella to replicate efficiently in vitro in high concentrations of ciprofloxacin. CONCLUSIONS: We found that below the CLSI breakpoint, there was no association between MIC and clinical outcome in paediatric shigellosis infections. However, S. flexneri patients had worse clinical outcomes when treated with gatifloxacin in this study regardless of MIC. Additionally, Shigella harbouring the qnrS gene are able to replicate efficiently in high concentrations of ciprofloxacin and we hypothesize that such strains possess a competitive advantage against fluoroquinolone-susceptible strains due to enhanced shedding and transmission.

Carrique-Mas JJ, Tue NT, Bryant JE, Saylors K, Cuong NV, Hoa NT, An NN, Hien VB, Lao PV, Tu NC et al. 2015. The baseline characteristics and interim analyses of the high-risk sentinel cohort of the Vietnam Initiative on Zoonotic InfectiONS (VIZIONS). Sci Rep, 5 (1), pp. 17965. | Show Abstract | Read more

The Vietnam Initiative for Zoonotic Infections (VIZIONS) includes community-based 'high-risk sentinel cohort' (HRSC) studies investigating individuals at risk of zoonotic infection due to occupational or residential exposure to animals. A total of 852 HRSC members were recruited between March 2013 and August 2014 from three provinces (Ha Noi, Dak Lak, and Dong Thap). The most numerous group (72.8%) corresponded to individuals living on farms, followed by slaughterers (16.3%) and animal health workers (8.5%). Nasal/pharyngeal and rectal swabs were collected from HRSC members at recruitment and after notifying illness. Exposure to exotic animals (including wild pigs, porcupine, monkey, civet, bamboo rat and bat) was highest for the Dak Lak cohort (53.7%), followed by Ha Noi (13.7%) and Dong Thap (4.0%). A total of 26.8% of individuals reported consumption of raw blood over the previous year; 33.6% slaughterers reported no use of protective equipment at work. Over 686 person-years of observation, 213 episodes of suspect infectious disease were notified, equivalent of 0.35 reports per person-year. Responsive samples were collected from animals in the farm cohort. There was noticeable time and space clustering of disease episodes suggesting that the VIZIONS set up is also suitable for the formal epidemiological investigation of disease outbreaks.

Van Dung N, Anh PH, Van Cuong N, Hoa NT, Carrique-Mas J, Hien VB, Sharp C, Rabaa M, Berto A, Campbell J et al. 2016. Large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in Vietnam. J Gen Virol, 97 (2), pp. 378-388. | Show Abstract | Read more

A recent survey of pigs in Dong Thap province, Vietnam identified a high frequency of enterovirus species G (EV-G) infection (144/198; 72.7%). Amongst these was a plethora of EV-G types (EV-G1, EV-G6 and four new types EV-G8-EV-G11). To better characterize the genetic diversity of EV-G and investigate the possible existence of further circulating types, we performed a larger-scale study on 484 pig and 45 farm-bred boar faecal samples collected in 2012 and 2014, respectively. All samples from the previous and current studies were also screened for kobuviruses. The overall EV infection frequency remained extremely high (395/484; 81.6%), but with comparable detection rates and viral loads between healthy and diarrhoeic pigs; this contrasted with less frequent detection of EV-G in boars (4/45; 8.9%). EV was most frequently detected in pigs ≤ 14 weeks old (∼ 95%) and declined in older pigs. Infections with EV-G1 and EV-G6 were most frequent, whilst less commonly detected types included EV-G3, EV-G4 and EV-G8-EV-G11, and five new types (EV-G12-EV-G16). In contrast, kobuvirus infection frequency was significantly higher in diarrhoeic pigs (40.9 versus 27.6%; P = 0.01). Kobuviruses also showed contrasting epizootiologies and age associations; a higher prevalence was found in boars (42%) compared with domestic pigs (29%), with the highest infection frequency amongst pigs >52 weeks old. Although genetically diverse, all kobuviruses identified belonged to the species Aichivirus C. In summary, this study confirms infection with EV-G was endemic in Vietnamese domestic pigs and exhibits high genetic diversity and extensive inter-type recombination.

Chung The H, Rabaa MA, Thanh DP, Ruekit S, Wangchuk S, Dorji T, Tshering KP, Nguyen TN, Vinh PV, Thanh TH et al. 2015. Introduction and establishment of fluoroquinolone-resistant Shigella sonnei into Bhutan. Microb Genom, 1 (6), pp. e000042. | Show Abstract | Read more

Shigella sonnei is a major contributor to the global burden of diarrhoeal disease, generally associated with dysenteric diarrhoea in developed countries but also emerging in developing countries. The reason for the recent success of S. sonnei is unknown, but is likely catalysed by its ability to acquire resistance against multiple antimicrobials. Between 2011 and 2013, S. sonnei exhibiting resistance to fluoroquinolones, the first-line treatment recommended for shigellosis, emerged in Bhutan. Aiming to reconstruct the introduction and establishment of fluoroquinolone-resistant S. sonnei populations in Bhutan, we performed whole-genome sequencing on 71 S. sonnei samples isolated in Bhutan between 2011 and 2013.We found that these strains represented an expansion of a clade within the previously described lineage III, found specifically in Central Asia. Temporal phylogenetic reconstruction demonstrated that all of the sequenced Bhutanese S. sonnei diverged from a single ancestor that was introduced into Bhutan around 2006. Our data additionally predicted that fluoroquinolone resistance, conferred by mutations in gyrA and parC, arose prior to the introduction of the founder strain into Bhutan. Once established in Bhutan, these S. sonnei had access to a broad gene pool, as indicated by the acquisition of extended-spectrum β-lactamase-encoding plasmids and genes encoding type IV pili. The data presented here outline a model for the introduction and maintenance of fluoroquinolone-resistant S. sonnei in a new setting. Given the current circulation of fluoroquinolone-resistant S. sonnei in Asia, we speculate that this pattern of introduction is being recapitulated across the region and beyond.

Nhung NT, Thuy CT, Trung NV, Campbell J, Baker S, Thwaites G, Hoa NT, Carrique-Mas J. 2015. Induction of Antimicrobial Resistance in Escherichia coli and Non-Typhoidal Salmonella Strains after Adaptation to Disinfectant Commonly Used on Farms in Vietnam. Antibiotics (Basel), 4 (4), pp. 480-494. | Show Abstract | Read more

In Vietnam, commercial disinfectants containing quaternary ammonium compounds (QACs) are commonly used in pig and poultry farms to maintain hygiene during production. We hypothesized that sustained exposure to sub-bactericidal concentrations of QAC-based disinfectants may result in increased levels of antimicrobial resistance (AMR) among Enterobacteriacea due to the increase of efflux pump expression. To test this hypothesis we exposed six antimicrobial-susceptible Escherichia coli (E. coli) and six antimicrobial-susceptible non-typhoidal Salmonella (NTS) isolates to increasing concentrations of a commonly used commercial disinfectant containing a mix of benzalkonium chloride and glutaraldehyde. Over the 12-day experiment, strains exhibited a significant change in their minimum inhibitory concentration (MIC) of the disinfectant product (mean increase of 31% (SD ± 40)) (p = 0.02, paired Wilcoxon test). Increases in MIC for the disinfectant product were strongly correlated with increases in MIC (or decreases in inhibition zone) for all antimicrobials (Pearson's correlation coefficient 0.71-0.83, all p < 0.01). The greatest increases in MIC (or decreases in inhibition zone) were observed for ampicillin, tetracycline, ciprofloxacin, and chloramphenicol, and the smallest for gentamicin, trimethoprim/sulphamethoxazole. The treatment of 155 representative E. coli isolates from farmed and wild animals in the Mekong Delta (Vietnam) with phenyl-arginine beta-naphthylamide (PAβN), a generic efflux pump inhibitor, resulted in reductions in the prevalence of AMR ranging from 0.7% to 3.3% in these organisms, indicating a small contribution of efflux pumps on the observed prevalence of AMR on farms. These results suggest that the mass usage of commercial disinfectants, many of which contain QACs, is potentially a contributing factor on the generation and maintenance of AMR in animal production in Vietnam.

Nichols C, Cruz Espinoza LM, von Kalckreuth V, Aaby P, Ahmed El Tayeb M, Ali M, Aseffa A, Bjerregaard-Andersen M, Breiman RF, Cosmas L et al. 2015. Bloodstream Infections and Frequency of Pretreatment Associated With Age and Hospitalization Status in Sub-Saharan Africa. Clin Infect Dis, 61 Suppl 4 (suppl 4), pp. S372-S379. | Show Abstract | Read more

BACKGROUND: The clinical diagnosis of bacterial bloodstream infections (BSIs) in sub-Saharan Africa is routinely confused with malaria due to overlapping symptoms. The Typhoid Surveillance in Africa Program (TSAP) recruited febrile inpatients and outpatients of all ages using identical study procedures and enrollment criteria, thus providing an opportunity to assess disease etiology and pretreatment patterns among children and adults. METHODS: Inpatients and outpatients of all ages with tympanic or axillary temperatures of ≥38.0 or ≥37.5°C, respectively, and inpatients only reporting fever within the previous 72 hours were eligible for recruitment. All recruited patients had one blood sample drawn and cultured for microorganisms. Data from 11 TSAP surveillance sites in nine different countries were used in the analysis. Bivariate analysis was used to compare frequencies of pretreatment and BSIs in febrile children (<15 years old) and adults (≥15 years old) in each country. Pooled Cochran Mantel-Haenszel odds ratios (ORs) were calculated for overall trends. RESULTS: There was no significant difference in the odds of a culture-proven BSI between children and adults among inpatients or outpatients. Among both inpatients and outpatients, children had significantly higher odds of having a contaminated blood culture compared with adults. Using country-pooled data, child outpatients had 66% higher odds of having Salmonella Typhi in their bloodstream than adults (OR, 1.66; 95% confidence interval [CI], 1.01-2.73). Overall, inpatient children had 59% higher odds of pretreatment with analgesics in comparison to inpatient adults (OR, 1.59; 95% CI, 1.28-1.97). CONCLUSIONS: The proportion of patients with culture-proven BSIs in children compared with adults was similar across the TSAP study population; however, outpatient children were more likely to have Salmonella Typhi infections than outpatient adults. This finding points to the importance of including outpatient facilities in surveillance efforts, particularly for the surveillance of typhoid fever. Strategies to reduce contamination among pediatric blood cultures are needed across the continent to prevent the misdiagnosis of BSI cases in children.

Rabaa MA, Tue NT, Phuc TM, Carrique-Mas J, Saylors K, Cotten M, Bryant JE, Nghia HD, Cuong NV, Pham HA et al. 2015. The Vietnam Initiative on Zoonotic Infections (VIZIONS): A Strategic Approach to Studying Emerging Zoonotic Infectious Diseases. Ecohealth, 12 (4), pp. 726-735. | Show Abstract | Read more

The effect of newly emerging or re-emerging infectious diseases of zoonotic origin in human populations can be potentially catastrophic, and large-scale investigations of such diseases are highly challenging. The monitoring of emergence events is subject to ascertainment bias, whether at the level of species discovery, emerging disease events, or disease outbreaks in human populations. Disease surveillance is generally performed post hoc, driven by a response to recent events and by the availability of detection and identification technologies. Additionally, the inventory of pathogens that exist in mammalian and other reservoirs is incomplete, and identifying those with the potential to cause disease in humans is rarely possible in advance. A major step in understanding the burden and diversity of zoonotic infections, the local behavioral and demographic risks of infection, and the risk of emergence of these pathogens in human populations is to establish surveillance networks in populations that maintain regular contact with diverse animal populations, and to simultaneously characterize pathogen diversity in human and animal populations. Vietnam has been an epicenter of disease emergence over the last decade, and practices at the human/animal interface may facilitate the likelihood of spillover of zoonotic pathogens into humans. To tackle the scientific issues surrounding the origins and emergence of zoonotic infections in Vietnam, we have established The Vietnam Initiative on Zoonotic Infections (VIZIONS). This countrywide project, in which several international institutions collaborate with Vietnamese organizations, is combining clinical data, epidemiology, high-throughput sequencing, and social sciences to address relevant one-health questions. Here, we describe the primary aims of the project, the infrastructure established to address our scientific questions, and the current status of the project. Our principal objective is to develop an integrated approach to the surveillance of pathogens circulating in both human and animal populations and assess how frequently they are exchanged. This infrastructure will facilitate systematic investigations of pathogen ecology and evolution, enhance understanding of viral cross-species transmission events, and identify relevant risk factors and drivers of zoonotic disease emergence.

Thompson CN, Zelner JL, Nhu TDOH, Phan MV, Hoang Le P, Nguyen Thanh H, Vu Thuy D, Minh Nguyen N, Ha Manh T, Van Hoang Minh T et al. 2015. The impact of environmental and climatic variation on the spatiotemporal trends of hospitalized pediatric diarrhea in Ho Chi Minh City, Vietnam. Health Place, 35 pp. 147-154. | Show Abstract | Read more

It is predicted that the integration of climate-based early warning systems into existing action plans will facilitate the timely provision of interventions to diarrheal disease epidemics in resource-poor settings. Diarrhea remains a considerable public health problem in Ho Chi Minh City (HCMC), Vietnam and we aimed to quantify variation in the impact of environmental conditions on diarrheal disease risk across the city. Using all inpatient diarrheal admissions data from three large hospitals within HCMC, we developed a mixed effects regression model to differentiate district-level variation in risk due to environmental conditions from the overarching seasonality of diarrheal disease hospitalization in HCMC. We identified considerable spatial heterogeneity in the risk of all-cause diarrhea across districts of HCMC with low elevation and differential responses to flooding, air temperature, and humidity driving further spatial heterogeneity in diarrheal disease risk. The incorporation of these results into predictive forecasting algorithms will provide a powerful resource to aid diarrheal disease prevention and control practices in HCMC and other similar settings.

Le V, Nhu NT, Cerdeno-Tarraga A, Campbell JI, Tuyen HT, Nhu TDOH, Tam PT, Schultsz C, Thwaites G, Thomson NR, Baker S. 2015. Genetic characterization of three qnrS1-harbouring multidrug-resistance plasmids and qnrS1-containing transposons circulating in Ho Chi Minh City, Vietnam. J Med Microbiol, 64 (8), pp. 869-878. | Show Abstract | Read more

Plasmid-mediated quinolone resistance (PMQR) refers to a family of closely related genes that confer decreased susceptibility to fluoroquinolones. PMQR genes are generally associated with integrons and/or plasmids that carry additional antimicrobial resistance genes active against a range of antimicrobials. In Ho Chi Minh City (HCMC), Vietnam, we have previously shown a high frequency of PMQR genes within commensal Enterobacteriaceae. However, there are limited available sequence data detailing the genetic context in which the PMQR genes reside, and a lack of understanding of how these genes spread across the Enterobacteriaceae. Here, we aimed to determine the genetic background facilitating the spread and maintenance of qnrS1, the dominant PMQR gene circulating in HCMC. We sequenced three qnrS1-carrying plasmids in their entirety to understand the genetic context of these qnrS1-embedded plasmids and also the association of qnrS1-mediated quinolone resistance with other antimicrobial resistance phenotypes. Annotation of the three qnrS1-containing plasmids revealed a qnrS1-containing transposon with a closely related structure. We screened 112 qnrS1-positive commensal Enterobacteriaceae isolated in the community and in a hospital in HCMC to detect the common transposon structure. We found the same transposon structure to be present in 71.4 % (45/63) of qnrS1-positive hospital isolates and in 36.7 % (18/49) of qnrS1-positive isolates from the community. The resulting sequence analysis of the qnrS1 environment suggested that qnrS1 genes are widely distributed and are mobilized on elements with a common genetic background. Our data add additional insight into mechanisms that facilitate resistance to multiple antimicrobials in Gram-negative bacteria in Vietnam.

Le Minh V, Thi Khanh Nhu N, Vinh Phat V, Thompson C, Huong Lan NP, Thieu Nga TV, Thanh Tam PT, Tuyen HT, Hoang Nhu TD, Van Hao N et al. 2015. In vitro activity of colistin in antimicrobial combination against carbapenem-resistant Acinetobacter baumannii isolated from patients with ventilator-associated pneumonia in Vietnam. J Med Microbiol, 64 (10), pp. 1162-1169. | Show Abstract | Read more

Acinetobacter baumannii has become one of the major infection threats in intensive care units (ICUs) globally. Since 2008, A. baumannii has been the leading cause of ventilator-associated pneumonia (VAP) in our ICU at an infectious disease hospital in southern Vietnam. The emergence of this pathogen in our setting is consistent with the persistence of a specific clone exhibiting resistance to carbapenems. Antimicrobial combinations may be a strategy to treat infections caused by these carbapenem-resistant A. baumannii. Therefore, we assessed potential antimicrobial combinations against local carbapenem-resistant A. baumannii by measuring in vitro interactions of colistin with four antimicrobials that are locally certified for treating VAP. We first performed antimicrobial susceptibility testing and multilocus variable number tandem repeat analysis (MLVA) genotyping on 74 A. baumannii isolated from quantitative tracheal aspirates from patients with VAP over an 18-month period. These 74 isolates could be subdivided into 21 main clusters by MLVA and >80 % were resistant to carbapenems. We selected 56 representative isolates for in vitro combination synergy testing. Synergy was observed in four (7 %), seven (13 %), 20 (36 %) and 38 (68 %) isolates with combinations of colistin with ceftazidime, ceftriaxone, imipenem and meropenem, respectively. Notably, more carbapenem-resistant A. baumannii isolates (36/43; 84 %) exhibited synergistic activity with a combination of colistin and meropenem than carbapenem-susceptible A. baumannii isolates (2/13; 15 %) (P = 0.023; Fisher's exact test). Our findings suggest that combinations of colistin and meropenem should be considered when treating carbapenem-resistant A. baumannii infections in Vietnam, and we advocate clinical trials investigating combination therapy for VAP.

Parry CM, Thieu NTV, Dolecek C, Karkey A, Gupta R, Turner P, Dance D, Maude RR, Ha V, Tran CN et al. 2015. Clinically and Microbiologically Derived Azithromycin Susceptibility Breakpoints for Salmonella enterica Serovars Typhi and Paratyphi A (vol 59, pg 2756, 2015) ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 59 (7), pp. 4364-4364. | Read more

Anh PH, Van Cuong N, Son NT, Tue NT, Kosoy M, Woolhouse ME, Baker S, Bryant JE, Thwaites G, Carrique-Mas JJ, Rabaa MA. 2015. Diversity of Bartonella spp. in Bats, Southern Vietnam. Emerg Infect Dis, 21 (7), pp. 1266-1267. | Read more

Parry CM, Thieu NT, Dolecek C, Karkey A, Gupta R, Turner P, Dance D, Maude RR, Ha V, Tran CN et al. 2015. Erratum for Parry et al., Clinically and microbiologically derived azithromycin susceptibility breakpoints for Salmonella enterica serovars Typhi and Paratyphi A. Antimicrob Agents Chemother, 59 (7), pp. 4364. | Read more

Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, Jenney A, Connor TR, Hsu LY, Severin J et al. 2015. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci U S A, 112 (27), pp. E3574-E3581. | Show Abstract | Read more

Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats.

Maude RR, de Jong HK, Wijedoru L, Fukushima M, Ghose A, Samad R, Hossain MA, Karim MR, Faiz MA, Parry CM, CMCH Typhoid Study Group. 2015. The diagnostic accuracy of three rapid diagnostic tests for typhoid fever at Chittagong Medical College Hospital, Chittagong, Bangladesh. Trop Med Int Health, 20 (10), pp. 1376-1384. | Show Abstract | Read more

OBJECTIVE: To determine the diagnostic accuracy of three rapid diagnostic tests (RDTs) for typhoid fever in febrile hospitalised patients in Bangladesh. METHODS: Febrile adults and children admitted to Chittagong Medical College Hospital, Bangladesh, were investigated with Bact/Alert(®) blood cultures and real-time PCR to detect Salmonella enterica Typhi and Paratyphi A and assays for Rickettsia, leptospirosis and dengue fever. Acute serum samples were examined with the LifeAssay (LA) Test-it™ Typhoid IgM lateral flow assay detecting IgM antibodies against S. Typhi O antigen, CTKBiotech Onsite Typhoid IgG/IgM Combo Rapid-test cassette lateral flow assay detecting IgG and IgM antibodies against S. Typhi O and H antigens and SD Bioline line assay for IgG and IgM antibodies against S. Typhi proteins. RESULTS: In 300 malaria smear-negative febrile patients [median (IQR) age of 13.5 (5-31) years], 34 (11.3%) had confirmed typhoid fever: 19 positive by blood culture for S. Typhi (three blood PCR positive) and 15 blood culture negative but PCR positive for S. Typhi in blood. The respective sensitivity and specificity of the three RDTs in patients using a composite reference standard of blood culture and/or PCR-confirmed typhoid fever were 59% and 61% for LifeAssay, 59% and 74% for the CTK IgM and/or IgG, and 24% and 96% for the SD Bioline RDT IgM and/or IgG. The LifeAssay RDT had a sensitivity of 63% and a specificity of 91% when modified with a positive cut-off of ≥2+ and analysed using a Bayesian latent class model. CONCLUSIONS: These typhoid RDTs demonstrated moderate diagnostic accuracies, and better tests are needed.

Le V, Khanh Nhu NT, Cerdeno-Tarraga A, Campbell JI, Tuyen HT, Hoang Nhu TD, Thanh Tam PT, Schultsz C, Thwaites G, Thomson NR, Baker S. 2015. The genetic characterization of three qnrS1 harbouring multi-drug resistance plasmids and qnrS1-containing transposons circulating in Ho Chi Minh City, Vietnam. J Med Microbiol, 64 (8), pp. 869-878. | Show Abstract | Read more

Plasmid-mediated quinolone resistance (PMQR) refers to a family of closely related genes that confer decreased susceptibility to fluoroquinolones. The PMQR genes are generally associated with integrons and/or plasmids that carry additional antimicrobial resistance genes active against a range of antimicrobials. In Ho Chi Minh City (HCMC) we have previously shown a high frequency of PMQR genes within commensal Enterobacteriaceae. However, there is limited available sequence data detailing the genetic context in which the PMQR genes reside, and a lack of understanding of how these genes spread across the Enterobacteriaceae. Here, we aimed to determine the genetic background facilitating the spread and maintenance of qnrS1, the dominant PMQR gene circulating in HCMC. We sequenced three qnrS1-carrying plasmids in their entirety to understand the genetic context of these qnrS1-embedded plasmids and also the association of qnrS1 mediated quinolone resistance with other antimicrobial resistance phenotypes. Annotation of the three qnrS1-containing plasmids revealed a qnrS1 containing transposon with a closely related structure. We screened 112 qnrS1 positive commensal Enterobacteriaceae isolated in the community and a hospital in HCMC to detect the common transposon structure. We found the same transposon structure to be present in 71.4% (45/63) of qnrS1 positive hospital isolates and in 36.7% (18/49) of qnrS1 positive isolates from the community. The resulting sequence analysis of the qnrS1 environment suggests that qnrS1 are widely distributed and are mobilised on elements with a common genetic background. Our data adds additional insight into mechanisms that facilitate resistance to multiple antimicrobials in Gram-negative bacteria in Vietnam.

Thompson CN, Duy PT, Baker S. 2015. The Rising Dominance of Shigella sonnei: An Intercontinental Shift in the Etiology of Bacillary Dysentery. PLoS Negl Trop Dis, 9 (6), pp. e0003708. | Show Abstract | Read more

Shigellosis is the major global cause of dysentery. Shigella sonnei, which has historically been more commonly isolated in developed countries, is undergoing an unprecedented expansion across industrializing regions in Asia, Latin America, and the Middle East. The precise reasons underpinning the epidemiological distribution of the various Shigella species and this global surge in S. sonnei are unclear but may be due to three major environmental pressures. First, natural passive immunization with the bacterium Plesiomonas shigelloides is hypothesized to protect populations with poor water supplies against S. sonnei. Improving the quality of drinking water supplies would, therefore, result in a reduction in P. shigelloides exposure and a subsequent reduction in environmental immunization against S. sonnei. Secondly, the ubiquitous amoeba species Acanthamoeba castellanii has been shown to phagocytize S. sonnei efficiently and symbiotically, thus allowing the bacteria access to a protected niche in which to withstand chlorination and other harsh environmental conditions in temperate countries. Finally, S. sonnei has emerged from Europe and begun to spread globally only relatively recently. A strong selective pressure from localized antimicrobial use additionally appears to have had a dramatic impact on the evolution of the S. sonnei population. We hypothesize that S. sonnei, which exhibits an exceptional ability to acquire antimicrobial resistance genes from commensal and pathogenic bacteria, has a competitive advantage over S. flexneri, particularly in areas with poorly regulated antimicrobial use. Continuing improvement in the quality of global drinking water supplies alongside the rapid development of antimicrobial resistance predicts the burden and international distribution of S. sonnei will only continue to grow. An effective vaccine against S. sonnei is overdue and may become one of our only weapons against this increasingly dominant and problematic gastrointestinal pathogen.

Wyres KL, Gorrie C, Edwards DJ, Wertheim HF, Hsu LY, Van Kinh N, Zadoks R, Baker S, Holt KE. 2015. Extensive Capsule Locus Variation and Large-Scale Genomic Recombination within the Klebsiella pneumoniae Clonal Group 258. Genome Biol Evol, 7 (5), pp. 1267-1279. | Show Abstract | Read more

Klebsiella pneumoniae clonal group (CG) 258, comprising sequence types (STs) 258, 11, and closely related variants, is associated with dissemination of the K. pneumoniae carbapenemase (KPC). Hospital outbreaks of KPC CG258 infections have been observed globally and are very difficult to treat. As a consequence, there is renewed interest in alternative infection control measures such as vaccines and phage or depolymerase treatments targeting the K. pneumoniae polysaccharide capsule. To date, 78 immunologically distinct capsule variants have been described in K. pneumoniae. Previous investigations of ST258 and a small number of closely related strains suggested that capsular variation was limited within this clone; only two distinct ST258 capsule polysaccharide synthesis (cps) loci have been identified, both acquired through large-scale recombination events (>50 kb). In contrast to previous studies, we report a comparative genomic analysis of the broader K. pneumoniae CG258 (n = 39). We identified 11 different cps loci within CG258, indicating that capsular switching is actually common within the complex. We observed several insertion sequences (IS) within the cps loci, and show further intraclone diversification of two cps loci through IS activity. Our data also indicate that several large-scale recombination events have shaped the genomes of CG258, and that definition of the complex should be broadened to include ST395 (also reported to harbor KPC). As only the second report of extensive intraclonal cps variation among Gram-negative bacterial species, our findings alter our understanding of the evolution of these organisms and have key implications for the design of control measures targeting K. pneumoniae capsules.

De Jong HK, Achouiti A, Koh GC, Parry CM, Baker S, Faiz MA, van Dissel JT, Vollaard AM, van Leeuwen EM, Roelofs JJ et al. 2015. Expression and function of S100A8/A9 (calprotectin) in human typhoid fever and the murine Salmonella model. PLoS Negl Trop Dis, 9 (4), pp. e0003663. | Show Abstract | Read more

BACKGROUND: Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model. METHODS AND PRINCIPAL FINDINGS: S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury. CONCLUSION: S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S. Typhimurium in mice.

Anders KL, Thompson CN, Thuy NT, Nguyet NM, Tu LETP, Dung TT, Phat VV, Van NT, Hieu NT, Tham NT et al. 2015. The epidemiology and aetiology of diarrhoeal disease in infancy in southern Vietnam: a birth cohort study. Int J Infect Dis, 35 pp. 3-10. | Show Abstract | Read more

OBJECTIVES: Previous studies indicate a high burden of diarrhoeal disease in Vietnamese children, however longitudinal community-based data on burden and aetiology are limited. The findings from a large, prospective cohort study of diarrhoeal disease in infants in southern Vietnam are presented herein. METHODS: Infants were enrolled at birth in urban Ho Chi Minh City and a semi-rural district in southern Vietnam, and followed for 12 months (n=6706). Diarrhoeal illness episodes were identified through clinic-based passive surveillance, hospital admissions, and self-reports. RESULTS: The minimum incidence of diarrhoeal illness in the first year of life was 271/1000 infant-years of observation for the whole cohort. Rotavirus was the most commonly detected pathogen (50% of positive samples), followed by norovirus (24%), Campylobacter (20%), Salmonella (18%), and Shigella (16%). Repeat infections were identified in 9% of infants infected with rotavirus, norovirus, Shigella, or Campylobacter, and 13% of those with Salmonella infections. CONCLUSIONS: The minimum incidence of diarrhoeal disease in infants in both urban and semi-rural settings in southern Vietnam was quantified prospectively. A large proportion of laboratory-diagnosed disease was caused by rotavirus and norovirus. These data highlight the unmet need for a rotavirus vaccine in Vietnam and provide evidence of the previously unrecognized burden of norovirus in infants.

Thompson CN, Phan MV, Hoang NV, Minh PV, Vinh NT, Thuy CT, Nga TT, Rabaa MA, Duy PT, Dung TT et al. 2015. A prospective multi-center observational study of children hospitalized with diarrhea in Ho Chi Minh City, Vietnam. Am J Trop Med Hyg, 92 (5), pp. 1045-1052. | Show Abstract | Read more

We performed a prospective multicenter study to address the lack of data on the etiology, clinical and demographic features of hospitalized pediatric diarrhea in Ho Chi Minh City (HCMC), Vietnam. Over 2,000 (1,419 symptomatic and 609 non-diarrheal control) children were enrolled in three hospitals over a 1-year period in 2009-2010. Aiming to detect a panel of pathogens, we identified a known diarrheal pathogen in stool samples from 1,067/1,419 (75.2%) children with diarrhea and from 81/609 (13.3%) children without diarrhea. Rotavirus predominated in the symptomatic children (664/1,419; 46.8%), followed by norovirus (293/1,419; 20.6%). The bacterial pathogens Salmonella, Campylobacter, and Shigella were cumulatively isolated from 204/1,419 (14.4%) diarrheal children and exhibited extensive antimicrobial resistance, most notably to fluoroquinolones and third-generation cephalosporins. We suggest renewed efforts in generation and implementation of policies to control the sale and prescription of antimicrobials to curb bacterial resistance and advise consideration of a subsidized rotavirus vaccination policy to limit the morbidity due to diarrheal disease in Vietnam.

Tu LT, Hoang NV, Cuong NV, Campbell J, Bryant JE, Hoa NT, Kiet BT, Thompson C, Duy DT, Phat VV et al. 2015. High levels of contamination and antimicrobial-resistant non-typhoidal Salmonella serovars on pig and poultry farms in the Mekong Delta of Vietnam. Epidemiol Infect, 143 (14), pp. 3074-3086. | Show Abstract | Read more

We investigated the prevalence, diversity, and antimicrobial resistance (AMR) profiles of non-typhoidal Salmonella (NTS) and associated risk factors on 341 pig, chicken, and duck farms in Dong Thap province (Mekong Delta, Vietnam). Sampling was stratified by species, district (four categories), and farm size (three categories). Pooled faeces, collected using boot swabs, were tested using ISO 6575: 2002 (Annex D). Isolates were serogrouped; group B isolates were tested by polymerase chain reaction to detect S. Typhimurium and (monophasic) serovar 4,[5],12:i:- variants. The farm-level adjusted NTS prevalence was 64·7%, 94·3% and 91·3% for chicken, duck and pig farms, respectively. Factors independently associated with NTS were duck farms [odds ratio (OR) 21·2], farm with >50 pigs (OR 11·9), pig farm with 5-50 pigs (OR 4·88) (vs. chickens), and frequent rodent sightings (OR 2·3). Both S. Typhimurium and monophasic S. Typhimurium were more common in duck farms. Isolates had a high prevalence of resistance (77·6%) against tetracycline, moderate resistance (20-30%) against chloramphenicol, sulfamethoxazole-trimethoprim, ampicillin and nalidixic acid, and low resistance (<5%) against ciprofloxacin and third-generation cephalosporins. Multidrug resistance (resistance against ⩾3 classes of antimicrobial) was independently associated with monophasic S. Typhimurium and other group B isolates (excluding S. Typhimurium) and pig farms. The unusually high prevalence of NTS on Mekong Delta farms poses formidable challenges for control.

Baker S. 2015. Infectious disease. A return to the pre-antimicrobial era? Science, 347 (6226), pp. 1064-1066. | Read more

Parry CM, Thieu NT, Dolecek C, Karkey A, Gupta R, Turner P, Dance D, Maude RR, Ha V, Tran CN et al. 2015. Clinically and microbiologically derived azithromycin susceptibility breakpoints for Salmonella enterica serovars Typhi and Paratyphi A. Antimicrob Agents Chemother, 59 (5), pp. 2756-2764. | Show Abstract | Read more

Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 μg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤ 16 μg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P < 0.001) for S. Paratyphi A. A zone size of ≥ 13 mm to a 5-μg azithromycin disk identified S. Typhi isolates with an MIC of ≤ 16 μg/ml with a sensitivity of 99.7%. An azithromycin MIC of ≤ 16 μg/ml or disk inhibition zone size of ≥ 13 mm enabled the detection of susceptible S. Typhi isolates that respond to azithromycin treatment. Further work is needed to define the response to treatment in S. Typhi isolates with an azithromycin MIC of >16 μg/ml and to determine MIC and disk breakpoints for S. Paratyphi A.

Thompson CN, Blacksell SD, Paris DH, Arjyal A, Karkey A, Dongol S, Giri A, Dolecek C, Day N, Baker S et al. 2015. Undifferentiated febrile illness in Kathmandu, Nepal. Am J Trop Med Hyg, 92 (4), pp. 875-878. | Show Abstract | Read more

Undifferentiated febrile illnesses (UFIs) are common in low- and middle-income countries. We prospectively investigated the causes of UFIs in 627 patients presenting to a tertiary referral hospital in Kathmandu, Nepal. Patients with microbiologically confirmed enteric fever (218 of 627; 34.8%) randomized to gatifloxacin or ofloxacin treatment were previously reported. We randomly selected 125 of 627 (20%) of these UFI patients, consisting of 96 of 409 (23%) cases with sterile blood cultures and 29 of 218 (13%) cases with enteric fever, for additional diagnostic investigations. We found serological evidence of acute murine typhus in 21 of 125 (17%) patients, with 12 of 21 (57%) patients polymerase chain reaction (PCR)-positive for Rickettsia typhi. Three UFI cases were quantitative PCR-positive for Rickettsia spp., two UFI cases were seropositive for Hantavirus, and one UFI case was seropositive for Q fever. Fever clearance time (FCT) for rickettsial infection was 44.5 hours (interquartile range = 26-66 hours), and there was no difference in FCT between ofloxacin or gatifloxacin. Murine typhus represents an important cause of predominantly urban UFIs in Nepal, and fluoroquinolones seem to be an effective empirical treatment.

Tu LTP, Hoang NVM, Cuong NV, Campbell J, Bryant JE, Hoa NT, Kiet BT, Thompson C, Duy DT, Phat VV et al. 2015. High levels of contamination and antimicrobial-resistant non-typhoidal Salmonella serovars on pig and poultry farms in the Mekong Delta of Vietnam Epidemiology and Infection, 143 (14), pp. 3074-3086. | Read more

Basnyat B, Baker S. 2015. Typhoid carriage in the gallbladder The Lancet, 386 (9998), pp. 1074. | Read more

Connor TR, Barker CR, Baker KS, Weill FX, Talukder KA, Smith AM, Baker S, Gouali M, Pham Thanh D, Jahan Azmi I et al. 2015. Species-wide whole genome sequencing reveals historical global spread and recent local persistence in Shigella flexneri. Elife, 4 (AUGUST2015), pp. e07335. | Show Abstract | Read more

Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence.




Baker KS, Dallman TJ, Ashton PM, Day M, Hughes G, Crook PD, Gilbart VL, Zittermann S, Allen VG, Howden BP et al. 2015. Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: A cross-sectional study The Lancet Infectious Diseases, 15 (8), pp. 913-921. | Read more

Chung The H, Karkey A, Pham Thanh D, Boinett CJ, Cain AK, Ellington M, Baker KS, Dongol S, Thompson C, Harris SR et al. 2015. A high-resolution genomic analysis of multidrug-resistant hospital outbreaks of Klebsiella pneumoniae. EMBO Mol Med, 7 (3), pp. 227-239. | Show Abstract | Read more

Multidrug-resistant (MDR) Klebsiella pneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic diversity of K. pneumoniae in resource-poor hospital settings. Through whole-genome sequencing (WGS), we reconstructed an outbreak of MDR K. pneumoniae occurring on high-dependency wards in a hospital in Kathmandu during 2012 with a case-fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae. Using phylogenetic reconstruction and lineage-specific PCR, our data predicted a scenario in which K. pneumoniae, circulating for 6 months before the outbreak, underwent a series of ward-specific clonal expansions after the acquisition of genes facilitating virulence and MDR. We suggest that the early detection of a specific NDM-1 containing lineage in 2011 would have alerted the high-dependency ward staff to intervene. We argue that some form of real-time genetic characterisation, alongside clade-specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high- and low-income settings.

Wong VK, Baker S, Pickard DJ, Parkhill J, Page AJ, Feasey NA, Kingsley RA, Thomson NR, Keane JA, Weill FX et al. 2015. Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events. Nat Genet, 47 (6), pp. 632-639. | Show Abstract | Read more

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.

Baker KS, Dallman TJ, Ashton PM, Day M, Hughes G, Crook PD, Gilbart VL, Zittermann S, Allen VG, Howden BP et al. 2015. Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: a cross-sectional study. Lancet Infect Dis, 15 (8), pp. 913-921. | Show Abstract | Read more

BACKGROUND: Shigellosis is an acute, severe bacterial colitis that, in high-income countries, is typically associated with travel to high-risk regions (Africa, Asia, and Latin America). Since the 1970s, shigellosis has also been reported as a sexually transmitted infection in men who have sex with men (MSM), in whom transmission is an important component of shigellosis epidemiology in high-income nations. We aimed to use sophisticated subtyping and international sampling to determine factors driving shigellosis emergence in MSM linked to an outbreak in the UK. METHODS: We did a large-scale, cross-sectional genomic epidemiological study of shigellosis cases collected from 29 countries between December, 1995, and June 8, 2014. Focusing on an ongoing epidemic in the UK, we collected and whole-genome sequenced clinical isolates of Shigella flexneri serotype 3a from high-risk and low-risk regions, including cases associated with travel and sex between men. We examined relationships between geographical, demographic, and clinical patient data with the isolate antimicrobial susceptibility, genetic data, and inferred evolutionary relationships. FINDINGS: We obtained 331 clinical isolates of S flexneri serotype 3a, including 275 from low-risk regions (44 from individuals who travelled to high-risk regions), 52 from high-risk regions, and four outgroup samples (ie, closely related, but genetically distinct isolates used to determine the root of the phylogenetic tree). We identified a recently emerged lineage of S flexneri 3a that has spread intercontinentally in less than 20 years throughout regions traditionally at low risk for shigellosis via sexual transmission in MSM. The lineage had acquired multiple antimicrobial resistance determinants, and prevailing sublineages were strongly associated with resistance to the macrolide azithromycin. Eight (4%) of 206 isolates from the MSM-associated lineage were obtained from patients who had previously provided an isolate; these serial isolations indicated atypical infection patterns (eg, reinfection). INTERPRETATION: We identified transmission-facilitating behaviours and atypical course(s) of infection as precipitating factors in shigellosis-affected MSM. The intercontinental spread of antimicrobial-resistant shigella through established transmission routes emphasises the need for new approaches to tackle the public health challenge of sexually transmitted infections in MSM. FUNDING: Wellcome Trust (grant number 098051).

Basnyat B, Baker S. 2015. Typhoid carriage in the gallbladder. Lancet, 386 (9998), pp. 1074. | Read more

Weinert LA, Chaudhuri RR, Wang J, Peters SE, Corander J, Jombart T, Baig A, Howell KJ, Vehkala M, Välimäki N et al. 2015. Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis. Nat Commun, 6 pp. 6740. | Show Abstract | Read more

Streptococcus suis causes disease in pigs worldwide and is increasingly implicated in zoonotic disease in East and South-East Asia. To understand the genetic basis of disease in S. suis, we study the genomes of 375 isolates with detailed clinical phenotypes from pigs and humans from the United Kingdom and Vietnam. Here, we show that isolates associated with disease contain substantially fewer genes than non-clinical isolates, but are more likely to encode virulence factors. Human disease isolates are limited to a single-virulent population, originating in the 1920, s when pig production was intensified, but no consistent genomic differences between pig and human isolates are observed. There is little geographical clustering of different S. suis subpopulations, and the bacterium undergoes high rates of recombination, implying that an increase in virulence anywhere in the world could have a global impact over a short timescale.

Van Cuong N, Carrique-Mas J, Vo Be H, An NN, Tue NT, Anh NL, Anh PH, Phuc NT, Baker S, Voutilainen L et al. 2015. Rodents and risk in the Mekong Delta of Vietnam: seroprevalence of selected zoonotic viruses in rodents and humans. Vector Borne Zoonotic Dis, 15 (1), pp. 65-72. | Show Abstract | Read more

In the Mekong Delta in southern Vietnam, rats are commonly traded in wet markets and sold live for food consumption. We investigated seroprevalence to selected groups of rodent-borne viruses among human populations with high levels of animal exposure and among co-located rodent populations. The indirect fluorescence antibody test (IFAT) was used to determine seropositivity to representative reference strains of hantaviruses (Dobrava virus [DOBV], Seoul virus [SEOV]), cowpox virus, arenaviruses (lymphocytic choriomeningitis virus [LCMV]), flaviviruses (tick-borne encephalitis virus [TBEV]), and rodent parechoviruses (Ljungan virus), using sera from 245 humans living in Dong Thap Province and 275 rodents representing the five common rodent species sold in wet markets and present in peridomestic and farm settings. Combined seropositivity to DOBV and SEOV among the rodents and humans was 6.9% (19/275) and 3.7% (9/245), respectively; 1.1% (3/275) and 4.5% (11/245) to cowpox virus; 5.4% (15/275) and 47.3% (116/245) for TBEV; and exposure to Ljungan virus was 18.8% (46/245) in humans, but 0% in rodents. Very little seroreactivity was observed to LCMV in either rodents (1/275, 0.4%) or humans (2/245, 0.8%). Molecular screening of rodent liver tissues using consensus primers for flaviviruses did not yield any amplicons, whereas molecular screening of rodent lung tissues for hantavirus yielded one hantavirus sequence (SEOV). In summary, these results indicate low to moderate levels of endemic hantavirus circulation, possible circulation of a flavivirus in rodent reservoirs, and the first available data on human exposures to parechoviruses in Vietnam. Although the current evidence suggests only limited exposure of humans to known rodent-borne diseases, further research is warranted to assess public health implications of the rodent trade.

Schreiber F, Kay S, Frankel G, Clare S, Goulding D, van de Vosse E, van Dissel JT, Strugnell R, Thwaites G, Kingsley RA et al. 2015. The Hd, Hj, and Hz66 flagella variants of Salmonella enterica serovar Typhi modify host responses and cellular interactions. Sci Rep, 5 (1), pp. 7947. | Show Abstract | Read more

Salmonella Typhi, the causative agent of typhoid fever, is a monophyletic, human-restricted bacterium that exhibits limited phenotypic variation. S. Typhi from Indonesia are a notable exception, with circulating strains expressing diverse flagella antigens including Hj, Hd and Hz66. Hypothesizing that S. Typhi flagella plays a key role during infection, we constructed an S. Typhi fliC mutant and otherwise isogenic S. Typhi strains expressing the Hj, Hd, Hz66 flagella antigens. Phenotyping revealed differences in flagellum structure, strain motility and immunogenicity, but not in the ability of flagellated isolates to induce TLR5 activity. Invasion assays using epithelial and macrophage cell lines revealed differences in the ability of these S. Typhi derivatives to invade cells or induce cellular restructuring in the form of ruffles. Notably, the Hj variant induced substantial ruffles that were not fully dependent on the GTPases that contribute to this process. These data highlight important differences in the phenotypic properties of S. Typhi flagella variation and how they impact on the pathogenesis of S. Typhi.

Thompson CN, Anders KL, Nhi LETQ, Tuyen HT, Van Minh P, Tu LETP, Nhu TDOH, Nhan NT, Ly TT, Duong VT et al. 2014. A cohort study to define the age-specific incidence and risk factors of Shigella diarrhoeal infections in Vietnamese children: a study protocol. BMC Public Health, 14 (1), pp. 1289. | Show Abstract | Read more

BACKGROUND: Shigella spp. are one of the most common causes of paediatric dysentery globally, responsible for a substantial proportion of diarrhoeal disease morbidity and mortality, particularly in industrialising regions. Alarming levels of antimicrobial resistance are now reported in S. flexneri and S. sonnei, hampering treatment options. Little is known, however, about the burden of infection and disease due to Shigella spp. in the community. METHODS/DESIGN: In order to estimate the incidence of this bacterial infection in the community in Ho Chi Minh City, Vietnam we have designed a longitudinal cohort to follow up approximately 700 children aged 12-60 months for two years with active and passive surveillance for diarrhoeal disease. Children will be seen at 6 month intervals for health checks where blood and stool samples will be collected. Families will also be contacted every two weeks for information on presence of diarrhoea in the child. Upon report of a diarrhoeal disease episode, study nurses will either travel to the family home to perform an evaluation or the family will attend a study hospital at a reduced cost, where a stool sample will also be collected. Case report forms collected at this time will detail information regarding disease history, risk factors and presence of disease in the household.Outcomes will include (i) age-specific incidence of Shigella spp. and other agents of diarrhoeal disease in the community, (ii) risk factors for identified aetiologies, (iii) rates of seroconversion to a host of gastrointestinal pathogens in the first few years of life. Further work regarding the longitudinal immune response to a variety of Shigella antigens, host genetics and candidate vaccine/diagnostic proteins will also be conducted. DISCUSSION: This is the largest longitudinal cohort with active surveillance designed specifically to investigate Shigella infection and disease. The study is strengthened by the active surveillance component, which will likely capture a substantial proportion of episodes not normally identified through passive or hospital-based surveillance. It is hoped that information from this study will aid in the design and implementation of Shigella vaccine trials in the future.

My PVT, Rabaa MA, Donato C, Cowley D, Phat VV, Dung TTN, Anh PH, Vinh H, Bryant JE, Kellam P et al. 2014. Novel porcine-like human G26P[19] rotavirus identified in hospitalized paediatric diarrhea patients in Ho Chi Minh City, Vietnam Journal of General Virology, 95 pp. 2727-2733. | Read more

Nhung NT, Cuong NV, Campbell J, Hoa NT, Bryant JE, Truc VN, Kiet BT, Jombart T, Trung NV, Hien VB et al. 2015. High levels of antimicrobial resistance among escherichia coli isolates from livestock farms and synanthropic rats and shrews in the Mekong Delta of Vietnam. Appl Environ Microbiol, 81 (3), pp. 812-820. | Show Abstract | Read more

In Mekong Delta farms (Vietnam), antimicrobials are extensively used, but limited data are available on levels of antimicrobial resistance (AMR) among Escherichia coli isolates. We performed a structured survey of AMR in E. coli isolates (n = 434) from 90 pig, chicken, and duck farms. The results were compared with AMR among E. coli isolates (n = 234) from 66 small wild animals (rats and shrews) trapped on farms and in forests and rice fields. The isolates were susceptibility tested against eight antimicrobials. E. coli isolates from farmed animals were resistant to a median of 4 (interquartile range [IQR], 3 to 6) antimicrobials versus 1 (IQR, 1 to 2) among wild mammal isolates (P < 0.001). The prevalences of AMR among farmed species isolates (versus wild animals) were as follows: tetracycline, 84.7% (versus 25.6%); ampicillin, 78.9% (versus 85.9%); trimethoprim-sulfamethoxazole, 52.1% (versus 18.8%); chloramphenicol, 39.9% (versus 22.5%); amoxicillin-clavulanic acid, 36.6% (versus 34.5%); and ciprofloxacin, 24.9% (versus 7.3%). The prevalence of multidrug resistance (MDR) (resistance against three or more antimicrobial classes) among pig isolates was 86.7% compared to 66.9 to 72.7% among poultry isolates. After adjusting for host species, MDR was ∼8 times greater among isolates from wild mammals trapped on farms than among those trapped in forests/rice fields (P < 0.001). Isolates were assigned to unique profiles representing their combinations of susceptibility results. Multivariable analysis of variance indicated that AMR profiles from wild mammals trapped on farms and those from domestic animals were more alike (R(2) range, 0.14 to 0.30) than E. coli isolates from domestic animals and mammals trapped in the wild (R(2) range, 0.25 to 0.45). The results strongly suggest that AMR on farms is a key driver of environmental AMR in the Mekong Delta.

My PV, Rabaa MA, Donato C, Cowley D, Phat VV, Dung TT, Anh PH, Vinh H, Bryant JE, Kellam P et al. 2014. Novel porcine-like human G26P[19] rotavirus identified in hospitalized paediatric diarrhoea patients in Ho Chi Minh City, Vietnam. J Gen Virol, 95 (Pt 12), pp. 2727-2733. | Show Abstract | Read more

During a hospital-based diarrhoeal disease study conducted in Ho Chi Minh City, Vietnam from 2009 to 2010, we identified four symptomatic children infected with G26P[19] rotavirus (RV)--an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P[19] strain, the whole genome of a representative example was characterized, revealing a novel genome constellation: G26-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine RVs (VP1-3 and NSP2-5). We proposed that this G26P[19] strain was the product of zoonotic transmission coupled with one or more reassortment events occurring in human and/or animal reservoirs. The identification of such strains has potential implications for vaccine efficacy in south-east Asia, and outlines the utility of whole-genome sequencing for studying RV diversity and zoonotic potential during disease surveillance.

Lan NP, Nga TV, Yen NT, Dung LET, Tuyen HT, Campbell JI, Whitehorn J, Thwaites G, Chau NV, Baker S. 2014. Two cases of bacteriemia caused by nontoxigenic, non-O1, non-O139 Vibrio cholerae isolates in Ho Chi Minh City, Vietnam. J Clin Microbiol, 52 (10), pp. 3819-3821. | Show Abstract | Read more

The toxigenic bacterium Vibrio cholerae belonging to the O1 and O139 serogroups is commonly associated with epidemic diarrhea in tropical settings; other diseases caused by this environmental pathogen are seldom identified. Here we report two unassociated cases of nonfatal, nontoxigenic V. cholerae non-O1, non-O139 bacteremia in patients with comorbidities in Ho Chi Minh City, Vietnam, that occurred within a 4-week period.

Thompson CN, Kama M, Acharya S, Bera U, Clemens J, Crump JA, Dawainavesi A, Dougan G, Edmunds WJ, Fox K et al. 2014. Typhoid fever in Fiji: a reversible plague? Trop Med Int Health, 19 (10), pp. 1284-1292. | Show Abstract | Read more

The country of Fiji, with a population of approximately 870 000 people, faces a growing burden of several communicable diseases including the bacterial infection typhoid fever. Surveillance data suggest that typhoid has become increasingly common in rural areas of Fiji and is more frequent amongst young adults. Transmission of the organisms that cause typhoid is facilitated by faecal contamination of food or water and may be influenced by local behavioural practices in Fiji. The Fijian Ministry of Health, with support from Australian Aid, hosted a meeting in August 2012 to develop comprehensive control and prevention strategies for typhoid fever in Fiji. International and local specialists were invited to share relevant data and discuss typhoid control options. The resultant recommendations focused on generating a clearer sense of the epidemiology of typhoid in Fiji and exploring the contribution of potential transmission pathways. Additionally, the panel suggested steps such as ensuring that recommended ciprofloxacin doses are appropriate to reduce the potential for relapse and reinfection in clinical cases, encouraging proper hand hygiene of food and drink handlers, working with water and sanitation agencies to review current sanitation practices and considering a vaccination policy targeting epidemiologically relevant populations.

Cotten M, Petrova V, Phan MV, Rabaa MA, Watson SJ, Ong SH, Kellam P, Baker S. 2014. Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting. J Virol, 88 (19), pp. 11056-11069. | Show Abstract | Read more

UNLABELLED: Norovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need, we have developed a whole-genome deep-sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all of the publically available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending three hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive-selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2), and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure. IMPORTANCE: The high transmissibility and rapid evolutionary rate of norovirus, combined with a short-lived host immune responses, are thought to be the reasons why the virus causes the majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have been described in detail for only a portion of the virus genome and never for a virus from a detailed urban tropical setting. We provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. This study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the method described here provides a robust full-genome sequencing platform for community-based virus surveillance.

Gunn JS, Marshall JM, Baker S, Dongol S, Charles RC, Ryan ET. 2014. Salmonella chronic carriage: epidemiology, diagnosis, and gallbladder persistence. Trends Microbiol, 22 (11), pp. 648-655. | Show Abstract | Read more

Typhoid (enteric fever) remains a major cause of morbidity and mortality worldwide, causing over 21 million new infections annually, with the majority of deaths occurring in young children. Because typhoid fever-causing Salmonella have no known environmental reservoir, the chronic, asymptomatic carrier state is thought to be a key feature of continued maintenance of the bacterium within human populations. Despite the importance of this disease to public health, our understanding of the molecular mechanisms that catalyze carriage, as well as our ability to reliably identify and treat the Salmonella carrier state, have only recently begun to advance.

Nhu NT, Lan NP, Campbell JI, Parry CM, Thompson C, Tuyen HT, Hoang NV, Tam PT, Le VM, Nga TV et al. 2014. Emergence of carbapenem-resistant Acinetobacter baumannii as the major cause of ventilator-associated pneumonia in intensive care unit patients at an infectious disease hospital in southern Vietnam. J Med Microbiol, 63 (Pt 10), pp. 1386-1394. | Show Abstract | Read more

Ventilator-associated pneumonia (VAP) is a serious healthcare-associated infection that affects up to 30 % of intubated and mechanically ventilated patients in intensive care units (ICUs) worldwide. The bacterial aetiology and corresponding antimicrobial susceptibility of VAP is highly variable, and can differ between countries, national provinces and even between different wards in the same hospital. We aimed to understand and document changes in the causative agents of VAP and their antimicrobial susceptibility profiles retrospectively over an 11 year period in a major infectious disease hospital in southern Vietnam. Our analysis outlined a significant shift from Pseudomonas aeruginosa to Acinetobacter spp. as the most prevalent bacteria isolated from quantitative tracheal aspirates in patients with VAP in this setting. Antimicrobial resistance was common across all bacterial species and we found a marked proportional annual increase in carbapenem-resistant Acinetobacter spp. over a 3 year period from 2008 (annual trend; odds ratio 1.656, P = 0.010). We further investigated the possible emergence of a carbapenem-resistant Acinetobacter baumannii clone by multiple-locus variable number tandem repeat analysis, finding a blaOXA-23-positive strain that was associated with an upsurge in the isolation of this pathogen. We additionally identified a single blaNDM-1-positive A. baumannii isolate. This work highlights the emergence of a carbapenem-resistant clone of A. baumannii and a worrying trend of antimicrobial resistance in the ICU of the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam.

Näsström E, Vu Thieu NT, Dongol S, Karkey A, Voong Vinh P, Ha Thanh T, Johansson A, Arjyal A, Thwaites G, Dolecek C et al. 2014. Salmonella Typhi and Salmonella Paratyphi A elaborate distinct systemic metabolite signatures during enteric fever. Elife, 3 | Show Abstract | Read more

The host-pathogen interactions induced by Salmonella Typhi and Salmonella Paratyphi A during enteric fever are poorly understood. This knowledge gap, and the human restricted nature of these bacteria, limit our understanding of the disease and impede the development of new diagnostic approaches. To investigate metabolite signals associated with enteric fever we performed two dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC/TOFMS) on plasma from patients with S. Typhi and S. Paratyphi A infections and asymptomatic controls, identifying 695 individual metabolite peaks. Applying supervised pattern recognition, we found highly significant and reproducible metabolite profiles separating S. Typhi cases, S. Paratyphi A cases, and controls, calculating that a combination of six metabolites could accurately define the etiological agent. For the first time we show that reproducible and serovar specific systemic biomarkers can be detected during enteric fever. Our work defines several biologically plausible metabolites that can be used to detect enteric fever, and unlocks the potential of this method in diagnosing other systemic bacterial infections.

Harrison JW, Dung TT, Siddiqui F, Korbrisate S, Bukhari H, Tra MP, Hoang NV, Carrique-Mas J, Bryant J, Campbell JI et al. 2014. Identification of possible virulence marker from Campylobacter jejuni isolates. Emerg Infect Dis, 20 (6), pp. 1026-1029. | Show Abstract | Read more

A novel protein translocation system, the type-6 secretion system (T6SS), may play a role in virulence of Campylobacter jejuni. We investigated 181 C. jejuni isolates from humans, chickens, and environmental sources in Vietnam, Thailand, Pakistan, and the United Kingdom for T6SS. The marker was most prevalent in human and chicken isolates from Vietnam.

Baker S, Karkey A, Parry C. 2014. Are we adequately prepared for the emergence of Salmonella enterica serovar Paratyphi A? Lancet Glob Health, 2 (4), pp. e195-e196. | Read more

Malla S, Dumre SP, Shakya G, Kansakar P, Rai B, Hossain A, Nair GB, Albert MJ, Sack D, Baker S et al. 2014. The challenges and successes of implementing a sustainable antimicrobial resistance surveillance programme in Nepal. BMC Public Health, 14 (1), pp. 269. | Show Abstract | Read more

BACKGROUND: Antimicrobial resistance (AMR) is a major global public health concern and its surveillance is a fundamental tool for monitoring the development of AMR. In 1998, the Nepalese Ministry of Health (MOH) launched an Infectious Disease (ID) programme. The key components of the programme were to establish a surveillance programme for AMR and to develop awareness among physicians regarding AMR and rational drug usage in Nepal. METHODS: An AMR surveillance programme was established and implemented by the Nepalese MOH in partnership with the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) from 1998 to 2003. From 2004 to 2012, the programme was integrated and maintained as a core activity of the National Public Health Laboratory (NPHL) and resulted in an increased number of participating laboratories and pathogens brought under surveillance. The main strategies were to build national capacity on isolation, identification and AMR testing of bacterial pathogens, establish laboratory networking and an External Quality Assessment (EQA) programme, promote standardised recording and reporting of results, and to ensure timely analysis and dissemination of data for advocacy and national policy adaptations. The programme was initiated by nine participating laboratories performing AMR surveillance on Vibrio cholerae, Shigella spp., Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. RESULTS: The number of participating laboratories was ultimately increased to 13 and the number of pathogens under surveillance was increased to seven (Salmonella spp. was added to the surveillance programme in 2002 and extended spectrum β-lactamase producing Escherichia coli in 2011). From 1999 to 2012, data were available on 17,103 bacterial isolates. During the AMR programme, we observed changing trends in serovars/species for Salmonella spp., Shigella spp. and V. cholerae and changing AMR trend for all organisms. Notably, N. gonorrhoeae isolates demonstrated increasing resistance to ciprofloxacin. Additionally, the performance of the participating laboratories improved as shown by annual EQA data evaluation. CONCLUSIONS: This Nepalese AMR programme continues and serves as a model for sustainable surveillance of AMR monitoring in resource limited settings.

Pham HA, Carrique-Mas JJ, Nguyen VC, Ngo TH, Nguyet LA, Do TD, Vo BH, Phan VT, Rabaa MA, Farrar J et al. 2014. The prevalence and genetic diversity of group A rotaviruses on pig farms in the Mekong Delta region of Vietnam. Vet Microbiol, 170 (3-4), pp. 258-265. | Show Abstract | Read more

Group A rotaviruses (ARoVs) are a common cause of severe diarrhea among children worldwide and the cause of approximately 45% of pediatric hospitalizations for acute diarrhea in Vietnam. ARoVs are known to cause significant economic losses to livestock producers by reducing growth performance and production efficiencies, however little is known about the implications of asymptomatic endemic circulation of ARoV. We aimed to determine the prevalence and predominant circulating genotypes of ARoVs on pig farms in a southern province of Vietnam. We found overall animal-level and farm-level prevalence of 32.7% (239/730) and 74% (77/104), respectively, and identified six different G types and 4 P types in various combinations (G2, G3, G4, G5, G9, G11 and P[6], P[13], P[23], and P[34]). There was no significant association between ARoV infection and clinical disease in pigs, suggesting that endemic asymptomatic circulation of ARoV may complicate rotavirus disease attribution during outbreaks of diarrhea in swine. Sequence analysis of the detected ARoVs suggested homology to recent human clinical cases and extensive genetic diversity. The epidemiological relevance of these findings for veterinary practitioners and to ongoing pediatric ARoV vaccine initiatives in Vietnam merits further study.

Charles RC, Liang L, Khanam F, Sayeed MA, Hung C, Leung DT, Baker S, Ludwig A, Harris JB, Larocque RC et al. 2014. Immunoproteomic analysis of antibody in lymphocyte supernatant in patients with typhoid fever in Bangladesh. Clin Vaccine Immunol, 21 (3), pp. 280-285. | Show Abstract | Read more

We have previously shown that an assay based on detection of anti-Salmonella enterica serotype Typhi antibodies in supernatant of lymphocytes harvested from patients presenting with typhoid fever (antibody in lymphocyte supernatant [ALS] assay) can identify 100% of patients with blood culture-confirmed typhoid fever in Bangladesh. In order to define immunodominant proteins within the S. Typhi membrane preparation used as antigen in these prior studies and to identify potential biomarkers unique to S. Typhi bacteremic patients, we probed microarrays containing 2,724 S. Typhi proteins with ALS collected at the time of clinical presentation from 10 Bangladeshis with acute typhoid fever. We identified 62 immunoreactive antigens when evaluating both the IgG and IgA responses. Immune responses to 10 of these antigens discriminated between individuals with acute typhoid infection and healthy control individuals from areas where typhoid infection is endemic, as well as Bangladeshi patients presenting with fever who were subsequently confirmed to have a nontyphoid illness. Using an ALS enzyme-linked immunosorbent assay (ELISA) format and purified antigen, we then confirmed that immune responses against the antigen with the highest immunoreactivity (hemolysin E [HlyE]) correctly identified individuals with acute typhoid or paratyphoid fever in Dhaka, Bangladesh. These observations suggest that purified antigens could be used with ALS and corresponding acute-phase activated B lymphocytes in diagnostic platforms to identify acutely infected patients, even in areas where enteric fever is endemic.

Gunn JS, Marshall JM, Baker S, Dongol S, Charles RC, Ryan ET. 2014. Salmonella chronic carriage: Epidemiology, diagnosis, and gallbladder persistence Trends in Microbiology, 22 (11), pp. 648-655. | Read more

Dunstan SJ, Hue NT, Han B, Li Z, Tram TT, Sim KS, Parry CM, Chinh NT, Vinh H, Lan NP et al. 2014. Variation at HLA-DRB1 is associated with resistance to enteric fever. Nat Genet, 46 (12), pp. 1333-1336. | Show Abstract | Read more

Enteric fever affects more than 25 million people annually and results from systemic infection with Salmonella enterica serovar Typhi or Paratyphi pathovars A, B or C(1). We conducted a genome-wide association study of 432 individuals with blood culture-confirmed enteric fever and 2,011 controls from Vietnam. We observed strong association at rs7765379 (odds ratio (OR) for the minor allele = 0.18, P = 4.5 × 10(-10)), a marker mapping to the HLA class II region, in proximity to HLA-DQB1 and HLA-DRB1. We replicated this association in 595 enteric fever cases and 386 controls from Nepal and also in a second independent collection of 151 cases and 668 controls from Vietnam. Imputation-based fine-mapping across the extended MHC region showed that the classical HLA-DRB1*04:05 allele (OR = 0.14, P = 2.60 × 10(-11)) could entirely explain the association at rs7765379, thus implicating HLA-DRB1 as a major contributor to resistance against enteric fever, presumably through antigen presentation.

Thompson CN, Kama M, Acharya S, Bera U, Clemens J, Crump JA, Dawainavesi A, Dougan G, Edmunds WJ, Fox K et al. 2014. Typhoid fever in Fiji: a reversible plague? Tropical medicine &amp; international health : TM &amp; IH, 19 (10), pp. 1284-1292. | Read more

Khauv P, Turner P, Soeng C, Soeng S, Moore CE, Bousfield R, Stoesser N, Emary K, Thanh DP, Baker S et al. 2014. Ophthalmic infections in children presenting to Angkor Hospital for Children, Siem Reap, Cambodia. BMC Res Notes, 7 (1), pp. 784. | Show Abstract | Read more

BACKGROUND: Ophthalmic infections cause significant morbidity in Cambodian children but aetiologic data are scarce. We investigated the causes of acute eye infections in 54 children presenting to the ophthalmology clinic at Angkor Hospital for Children, Siem Reap between March and October 2012. FINDINGS: The median age at presentation was 3.6 years (range 6 days - 16.0 years). Forty two patients (77.8%) were classified as having an external eye infection, ten (18.5%) as ophthalmia neonatorum, and two (3.7%) as intra-ocular infection. Organisms were identified in all ophthalmia neonatorum patients and 85.7% of patients with an external eye infection. Pathogens were not detected in either of the intra-ocular infection patients. Most commonly isolated bacteria were Staphylococcus aureus (23 isolates), coagulase-negative staphylococci (13), coliforms (7), Haemophilus influenzae/parainfluenzae (6), Streptococcus pneumoniae (4), and Neisseria gonorrhoeae (2). Chlamydia trachomatis DNA was detected in 60% of swabs taken from ophthalmia neonatorum cases. CONCLUSIONS: This small study demonstrates the wide range of pathogens associated with common eye infections in Cambodian children. The inclusion of molecular assays improved the spectrum of detectable pathogens, most notably in neonates.

Pitzer VE, Bowles CC, Baker S, Kang G, Balaji V, Farrar JJ, Grenfell BT. 2014. Predicting the impact of vaccination on the transmission dynamics of typhoid in South Asia: a mathematical modeling study. PLoS neglected tropical diseases, 8 (1), | Read more

Hong Anh P, Carrique-Mas JJ, Van Cuong N, Hoa NT, Lam Anh N, Duy DT, Hien VB, Vu Tra My P, Rabaa MA, Farrar J et al. 2014. The prevalence and genetic diversity of group A rotaviruses on pig farms in the Mekong Delta region of Vietnam Veterinary Microbiology, 170 (3-4), pp. 258-265. | Read more

Parry CM, Thompson C, Vinh H, Chinh NT, Phuong LET, Ho VA, Hien TT, Wain J, Farrar JJ, Baker S. 2014. Risk factors for the development of severe typhoid fever in Vietnam. BMC Infect Dis, 14 (1), pp. 73. | Show Abstract | Read more

BACKGROUND: Typhoid fever is a systemic infection caused by the bacterium Salmonella enterica serovar Typhi. Age, sex, prolonged duration of illness, and infection with an antimicrobial resistant organism have been proposed risk factors for the development of severe disease or fatality in typhoid fever. METHODS: We analysed clinical data from 581 patients consecutively admitted with culture confirmed typhoid fever to two hospitals in Vietnam during two periods in 1993-1995 and 1997-1999. These periods spanned a change in the antimicrobial resistance phenotypes of the infecting organisms i.e. fully susceptible to standard antimicrobials, resistance to chloramphenicol, ampicillin and trimethoprim-sulphamethoxazole (multidrug resistant, MDR), and intermediate susceptibility to ciprofloxacin (nalidixic acid resistant). Age, sex, duration of illness prior to admission, hospital location and the presence of MDR or intermediate ciprofloxacin susceptibility in the infecting organism were examined by logistic regression analysis to identify factors independently associated with severe typhoid at the time of hospital admission. RESULTS: The prevalence of severe typhoid was 15.5% (90/581) and included: gastrointestinal bleeding (43; 7.4%); hepatitis (29; 5.0%); encephalopathy (16; 2.8%); myocarditis (12; 2.1%); intestinal perforation (6; 1.0%); haemodynamic shock (5; 0.9%), and death (3; 0.5%). Severe disease was more common with increasing age, in those with a longer duration of illness and in patients infected with an organism exhibiting intermediate susceptibility to ciprofloxacin. Notably an MDR phenotype was not associated with severe disease. Severe disease was independently associated with infection with an organism with an intermediate susceptibility to ciprofloxacin (AOR 1.90; 95% CI 1.18-3.07; p = 0.009) and male sex (AOR 1.61 (1.00-2.57; p = 0.035). CONCLUSIONS: In this group of patients hospitalised with typhoid fever infection with an organism with intermediate susceptibility to ciprofloxacin was independently associated with disease severity. During this period many patients were being treated with fluoroquinolones prior to hospital admission. Ciprofloxacin and ofloxacin should be used with caution in patients infected with S. Typhi that have intermediate susceptibility to ciprofloxacin.

Pitzer VE, Bowles CC, Baker S, Kang G, Balaji V, Farrar JJ, Grenfell BT. 2014. Predicting the impact of vaccination on the transmission dynamics of typhoid in South Asia: a mathematical modeling study. PLoS Negl Trop Dis, 8 (1), pp. e2642. | Show Abstract | Read more

BACKGROUND: Modeling of the transmission dynamics of typhoid allows for an evaluation of the potential direct and indirect effects of vaccination; however, relevant typhoid models rooted in data have rarely been deployed. METHODOLOGY/PRINCIPAL FINDINGS: We developed a parsimonious age-structured model describing the natural history and immunity to typhoid infection. The model was fit to data on culture-confirmed cases of typhoid fever presenting to Christian Medical College hospital in Vellore, India from 2000-2012. The model was then used to evaluate the potential impact of school-based vaccination strategies using live oral, Vi-polysaccharide, and Vi-conjugate vaccines. The model was able to reproduce the incidence and age distribution of typhoid cases in Vellore. The basic reproductive number (R 0) of typhoid was estimated to be 2.8 in this setting. Vaccination was predicted to confer substantial indirect protection leading to a decrease in the incidence of typhoid in the short term, but (intuitively) typhoid incidence was predicted to rebound 5-15 years following a one-time campaign. CONCLUSIONS/SIGNIFICANCE: We found that model predictions for the overall and indirect effects of vaccination depend strongly on the role of chronic carriers in transmission. Carrier transmissibility was tentatively estimated to be low, consistent with recent studies, but was identified as a pivotal area for future research. It is unlikely that typhoid can be eliminated from endemic settings through vaccination alone.

Shah MA, Mutreja A, Thomson N, Baker S, Parkhill J, Dougan G, Bokhari H, Wren BW. 2014. Genomic epidemiology of Vibrio cholerae O1 associated with floods, Pakistan, 2010. Emerg Infect Dis, 20 (1), pp. 13-20. | Show Abstract | Read more

In August 2010, Pakistan experienced major floods and a subsequent cholera epidemic. To clarify the population dynamics and transmission of Vibrio cholerae in Pakistan, we sequenced the genomes of all V. cholerae O1 El Tor isolates and compared the sequences to a global collection of 146 V. cholerae strains. Within the global phylogeny, all isolates from Pakistan formed 2 new subclades (PSC-1 and PSC-2), lying in the third transmission wave of the seventh-pandemic lineage that could be distinguished by signature deletions and their antimicrobial susceptibilities. Geographically, PSC-1 isolates originated from the coast, whereas PSC-2 isolates originated from inland areas flooded by the Indus River. Single-nucleotide polymorphism accumulation analysis correlated river flow direction with the spread of PSC-2. We found at least 2 sources of cholera in Pakistan during the 2010 epidemic and illustrate the value of a global genomic data bank in contextualizing cholera outbreaks.

Baker S, Duy PT, Nga TVT, Dung TTN, Phat VV, Chau TT, Keith Turner A, Farrar J, Boni MF. 2013. Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobial pressure eLife, 2013 (2), | Read more

Van Dung N, Anh PH, Van Cuong N, Hoa NT, Carrique-Mas J, Hien VB, Campbell J, Baker S, Farrar J, Woolhouse ME et al. 2014. Prevalence, genetic diversity and recombination of species G enteroviruses infecting pigs in Vietnam. J Gen Virol, 95 (Pt 3), pp. 549-556. | Show Abstract | Read more

Picornaviruses infecting pigs, described for many years as 'porcine enteroviruses', have recently been recognized as distinct viruses within three distinct genera (Teschovirus, Sapelovirus and Enterovirus). To better characterize the epidemiology and genetic diversity of members of the Enterovirus genus, faecal samples from pigs from four provinces in Vietnam were screened by PCR using conserved enterovirus (EV)-specific primers from the 5' untranslated region (5' UTR). High rates of infection were recorded in pigs on all farms, with detection frequencies of approximately 90% in recently weaned pigs but declining to 40% in those aged over 1 year. No differences in EV detection rates were observed between pigs with and without diarrhoea [74% (n = 70) compared with 72% (n = 128)]. Genetic analysis of consensus VP4/VP2 and VP1 sequences amplified from a subset of EV-infected pigs identified species G EVs in all samples. Among these, VP1 sequence comparisons identified six type 1 and seven type 6 variants, while four further VP1 sequences failed to group with any previously identified EV-G types. These have now been formally assigned as EV-G types 8-11 by the Picornavirus Study Group. Comparison of VP1, VP4/VP2, 3D(pol) and 5' UTRs of study samples and those available on public databases showed frequent, bootstrap-supported differences in their phylogenies indicative of extensive within-species recombination between genome regions. In summary, we identified extremely high frequencies of infection with EV-G in pigs in Vietnam, substantial genetic diversity and recombination within the species, and evidence for a much larger number of circulating EV-G types than currently described.

Shieh M, Thompson C, Phan Vu Tra M, Thi Thuy Linh V, Tediosi F, Merson L, Farrar JJ, Manh Tuan H, Lu Viet H, Thi Ngoc Tuyet P, Baker S. 2013. The policy of free healthcare for children under the age of 6 years in Vietnam: Assessment of the uptake for children hospitalised with acute diarrhoea in Ho Chi Minh City Tropical Medicine and International Health, 18 (12), pp. 1444-1451. | Read more

Moore CE, Pan-Ngum W, Wijedoru LP, Sona S, Nga TV, Duy PT, Vinh PV, Chheng K, Kumar V, Emary K et al. 2014. Evaluation of the diagnostic accuracy of a typhoid IgM flow assay for the diagnosis of typhoid fever in Cambodian children using a Bayesian latent class model assuming an imperfect gold standard. Am J Trop Med Hyg, 90 (1), pp. 114-120. | Show Abstract | Read more

Rapid diagnostic tests are needed for typhoid fever (TF) diagnosis in febrile children in endemic areas. Five hundred children admitted to the hospital in Cambodia between 2009 and 2010 with documented fever (≥ 38°C) were investigated using blood cultures (BCs), Salmonella Typhi/Paratyphi A real-time polymerase chain reactions (PCRs), and a Typhoid immunoglobulin M flow assay (IgMFA). Test performance was determined by conventional methods and Bayesian latent class modeling. There were 32 cases of TF (10 BC- and PCR-positive cases, 14 BC-positive and PCR-negative cases, and 8 BC-negative and PCR-positive cases). IgMFA sensitivity was 59.4% (95% confidence interval = 41-76), and specificity was 97.8% (95% confidence interval = 96-99). The model estimate sensitivity for BC was 81.0% (95% credible interval = 54-99). The model estimate sensitivity for PCR was 37.8% (95% credible interval = 26-55), with a specificity of 98.2% (95% credible interval = 97-99). The model estimate sensitivity for IgMFA (≥ 2+) was 77.9% (95% credible interval = 58-90), with a specificity of 97.5% (95% credible interval = 95-100). The model estimates of IgMFA sensitivity and specificity were comparable with BCs and better than estimates using conventional analysis.

Holt KE, Thieu Nga TV, Thanh DP, Vinh H, Kim DW, Vu Tra MP, Campbell JI, Hoang NV, Vinh NT, Minh PV et al. 2013. Tracking the establishment of local endemic populations of an emergent enteric pathogen. Proc Natl Acad Sci U S A, 110 (43), pp. 17522-17527. | Show Abstract | Read more

Shigella sonnei is a human-adapted pathogen that is emerging globally as the dominant agent of bacterial dysentery. To investigate local establishment, we sequenced the genomes of 263 Vietnamese S. sonnei isolated over 15 y. Our data show that S. sonnei was introduced into Vietnam in the 1980s and has undergone localized clonal expansion, punctuated by genomic fixation events through periodic selective sweeps. We uncover geographical spread, spatially restricted frontier populations, and convergent evolution through local gene pool sampling. This work provides a unique, high-resolution insight into the microevolution of a pioneering human pathogen during its establishment in a new host population.

Carrique-Mas JJ, Bryant JE, Cuong NV, Hoang NV, Campbell J, Hoang NV, Dung TT, Duy DT, Hoa NT, Thompson C et al. 2014. An epidemiological investigation of Campylobacter in pig and poultry farms in the Mekong delta of Vietnam. Epidemiol Infect, 142 (7), pp. 1425-1436. | Show Abstract | Read more

Campylobacter are zoonotic pathogens commonly associated with gastroenteritis. To assess the relevance of Campylobacter in Vietnam, an economically transitioning country in SE Asia, we conducted a survey of 343 pig and poultry farms in the Mekong delta, a region characterized by mixed species farming with limited biosecurity. The animal-level prevalence of Campylobacter was 31·9%, 23·9% and 53·7% for chickens, ducks and pigs, respectively. C. jejuni was predominant in all three host species, with the highest prevalence in pigs in high-density production areas. Campylobacter isolates demonstrated high levels of antimicrobial resistance (21% and 100% resistance against ciprofloxacin and erythromycin, respectively). Multilocus sequence type genotyping showed a high level of genetic diversity within C. jejuni, and predicted C. coli inter-species transmission. We suggest that on-going intensification of animal production systems, limited biosecurity, and increased urbanization in Vietnam is likely to result in Campylobacter becoming an increasingly significant cause of human diarrhoeal infections in coming years.

Charles RC, Sultana T, Alam MM, Yu Y, Wu-Freeman Y, Bufano MK, Rollins SM, Tsai L, Harris JB, LaRocque RC et al. 2013. Identification of immunogenic Salmonella enterica serotype Typhi antigens expressed in chronic biliary carriers of S. Typhi in Kathmandu, Nepal. PLoS Negl Trop Dis, 7 (8), pp. e2335. | Show Abstract | Read more

BACKGROUND: Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage. METHODOLOGY/PRINCIPAL FINDINGS: To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT), to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479), an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70%) chronic carriers, 0 of 8 bile culture-negative controls (0%), 0 of 8 healthy Bangladeshis (0%), and 1 of 8 (12.5%) Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present. CONCLUSIONS/SIGNIFICANCE: Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic S. Typhi carriers.

Pham Thanh D, Tran Vu Thieu N, Tran Thuy C, Lodén M, Tuin K, Campbell JI, Van Minh Hoang N, Voong Vinh P, Farrar JJ, Holt KE et al. 2013. Identification of Salmonella enterica serovar Typhi genotypes by use of rapid multiplex ligation-dependent probe amplification. J Clin Microbiol, 51 (9), pp. 2950-2958. | Show Abstract | Read more

Salmonella enterica serovar Typhi, the causative agent of typhoid fever, is highly clonal and genetically conserved, making isolate subtyping difficult. We describe a standardized multiplex ligation-dependent probe amplification (MLPA) genotyping scheme targeting 11 key phylogenetic markers of the S. Typhi genome. The MLPA method demonstrated 90% concordance with single nucleotide polymorphism (SNP) typing, the gold standard for S. Typhi genotyping, and had the ability to identify isolates of the H58 haplotype, which is associated with resistance to multiple antimicrobials. Additionally, the assay permitted the detection of fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA and the topoisomerase gene parC with a sensitivity of 100%. The MLPA methodology is simple and reliable, providing phylogenetically and phenotypically relevant genotyping information. This MLPA scheme offers a more-sensitive and interpretable alternative to the nonphylogenetic subgrouping methodologies that are currently used in reference and research laboratories in areas where typhoid is endemic.

Andrews JR, Prajapati KG, Eypper E, Shrestha P, Shakya M, Pathak KR, Joshi N, Tiwari P, Risal M, Koirala S et al. 2013. Evaluation of an electricity-free, culture-based approach for detecting typhoidal Salmonella bacteremia during enteric fever in a high burden, resource-limited setting. PLoS Negl Trop Dis, 7 (6), pp. e2292. | Show Abstract | Read more

BACKGROUND: In many rural areas at risk for enteric fever, there are few data on Salmonella enterica serotypes Typhi (S. Typhi) and Paratyphi (S. Paratyphi) incidence, due to limited laboratory capacity for microbiologic culture. Here, we describe an approach that permits recovery of the causative agents of enteric fever in such settings. This approach involves the use of an electricity-free incubator based upon use of phase-change materials. We compared this against conventional blood culture for detection of typhoidal Salmonella. METHODOLOGY/PRINCIPAL FINDINGS: Three hundred and four patients with undifferentiated fever attending the outpatient and emergency departments of a public hospital in the Kathmandu Valley of Nepal were recruited. Conventional blood culture was compared against an electricity-free culture approach. Blood from 66 (21.7%) patients tested positive for a Gram-negative bacterium by at least one of the two methods. Sixty-five (21.4%) patients tested blood culture positive for S. Typhi (30; 9.9%) or S. Paratyphi A (35; 11.5%). From the 65 individuals with culture-confirmed enteric fever, 55 (84.6%) were identified by the conventional blood culture and 60 (92.3%) were identified by the experimental method. Median time-to-positivity was 2 days for both procedures. The experimental approach was falsely positive due to probable skin contaminants in 2 of 239 individuals (0.8%). The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% (95% CI: 80.0%-97.0%) and 96.0% (92.7%-98.1%), respectively. After initial incubation, Salmonella isolates could be readily recovered from blood culture bottles maintained at room temperature for six months. CONCLUSIONS/SIGNIFICANCE: A simple culture approach based upon a phase-change incubator can be used to isolate agents of enteric fever. This approach could be used as a surveillance tool to assess incidence and drug resistance of the etiologic agents of enteric fever in settings without reliable local access to electricity or local diagnostic microbiology laboratories.

My PV, Thompson C, Phuc HL, Tuyet PT, Vinh H, Hoang NV, Minh PV, Vinh NT, Thuy CT, Nga TT et al. 2013. Endemic norovirus infections in children, Ho Chi Minh City, Vietnam, 2009-2010. Emerg Infect Dis, 19 (6), pp. 977-980. | Show Abstract | Read more

We performed a case-control investigation to identify risk factors for norovirus infections among children in Vietnam. Of samples from 1,419 children who had diarrhea and 609 who were asymptomatic, 20.6% and 2.8%, respectively, were norovirus positive. Risk factors included residential crowding and symptomatic contacts, indicating person-to-person transmission of norovirus.

Seth-Smith HM, Harris SR, Skilton RJ, Radebe FM, Golparian D, Shipitsyna E, Duy PT, Scott P, Cutcliffe LT, O'Neill C et al. 2013. Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture. Genome Res, 23 (5), pp. 855-866. | Show Abstract | Read more

The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C. trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C. trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C. trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.

Tra My PV, Lam HM, Thompson CN, Phuc HL, Tuyet PT, Vinh H, Hoang NV, Minh P, Vinh NT, Thuy CT et al. 2013. The dynamics of GII.4 Norovirus in Ho Chi Minh City, Vietnam. Infect Genet Evol, 18 pp. 335-343. | Show Abstract | Read more

Norovirus (NoV) is a major cause of epidemic gastroenteritis in industrialized countries, yet the epidemiological significance of NoV in industrializing countries remains poorly understood. The spatiotemporal distribution of NoV genotypes identified in 2054 enrolled children was investigated between May 2009 and December 2010, in Ho Chi Minh City (HCMC), Vietnam. A total of 315 NoV extracted from stool samples were genotyped and GPS mapped to their source. Genogroup II NoV, particularly GII.4, were predominant, and the GII.4 strains could be subgrouped into GII.4-2006b (Minerva) and GII.4-2010 (New Orleans) variants. There was no spatiotemporal structure among the endemic GII strains; yet a significant spatiotemporal signal corresponding with the novel introduction of GII.4-2010 variant was detected. These data show that NoV GII.4 variants are highly endemic in HCMC and describe a scenario of rapid NoV strain replacement occurring in HCMC in early 2010.

Day JN, Chau TT, Wolbers M, Mai PP, Dung NT, Mai NH, Phu NH, Nghia HD, Phong ND, Thai CQ et al. 2013. Combination antifungal therapy for cryptococcal meningitis. N Engl J Med, 368 (14), pp. 1291-1302. | Show Abstract | Read more

BACKGROUND: Combination antifungal therapy (amphotericin B deoxycholate and flucytosine) is the recommended treatment for cryptococcal meningitis but has not been shown to reduce mortality, as compared with amphotericin B alone. We performed a randomized, controlled trial to determine whether combining flucytosine or high-dose fluconazole with high-dose amphotericin B improved survival at 14 and 70 days. METHODS: We conducted a randomized, three-group, open-label trial of induction therapy for cryptococcal meningitis in patients with human immunodeficiency virus infection. All patients received amphotericin B at a dose of 1 mg per kilogram of body weight per day; patients in group 1 were treated for 4 weeks, and those in groups 2 and 3 for 2 weeks. Patients in group 2 concurrently received flucytosine at a dose of 100 mg per kilogram per day for 2 weeks, and those in group 3 concurrently received fluconazole at a dose of 400 mg twice daily for 2 weeks. RESULTS: A total of 299 patients were enrolled. Fewer deaths occurred by days 14 and 70 among patients receiving amphotericin B and flucytosine than among those receiving amphotericin B alone (15 vs. 25 deaths by day 14; hazard ratio, 0.57; 95% confidence interval [CI], 0.30 to 1.08; unadjusted P=0.08; and 30 vs. 44 deaths by day 70; hazard ratio, 0.61; 95% CI, 0.39 to 0.97; unadjusted P=0.04). Combination therapy with fluconazole had no significant effect on survival, as compared with monotherapy (hazard ratio for death by 14 days, 0.78; 95% CI, 0.44 to 1.41; P=0.42; hazard ratio for death by 70 days, 0.71; 95% CI, 0.45 to 1.11; P=0.13). Amphotericin B plus flucytosine was associated with significantly increased rates of yeast clearance from cerebrospinal fluid (-0.42 log10 colony-forming units [CFU] per milliliter per day vs. -0.31 and -0.32 log10 CFU per milliliter per day in groups 1 and 3, respectively; P<0.001 for both comparisons). Rates of adverse events were similar in all groups, although neutropenia was more frequent in patients receiving a combination therapy. CONCLUSIONS: Amphotericin B plus flucytosine, as compared with amphotericin B alone, is associated with improved survival among patients with cryptococcal meningitis. A survival benefit of amphotericin B plus fluconazole was not found. (Funded by the Wellcome Trust and the British Infection Society; number, ISRCTN95123928.).

Hoa NT, Chieu TT, Do Dung S, Long NT, Hieu TQ, Luc NT, Nhuong PT, Huong VT, Trinh DT, Wertheim HF et al. 2013. Streptococcus suis and porcine reproductive and respiratory syndrome, Vietnam. Emerg Infect Dis, 19 (2), pp. 331-333. | Read more

Kolader ME, Vinh H, Ngoc Tuyet PT, Thompson C, Wolbers M, Merson L, Campbell JI, Ngoc Dung TT, Manh Tuan H, Vinh Chau NV et al. 2013. An oral preparation of Lactobacillus acidophilus for the treatment of uncomplicated acute watery diarrhoea in Vietnamese children: study protocol for a multicentre, randomised, placebo-controlled trial. Trials, 14 (1), pp. 27. | Show Abstract | Read more

BACKGROUND: Diarrhoeal disease is a major global health problem, particularly affecting children under the age of 5 years. Besides oral rehydration solution, probiotics are also commonly prescribed to children with acute watery diarrhoea in some settings. Results from randomised clinical trials (RCTs) in which investigators studied the effect of probiotics on diarrhoeal symptoms have largely shown a positive effect; yet, the overall quality of the data is limited. In Vietnam, probiotics are the most frequently prescribed treatment for children hospitalised with acute watery diarrhoea, but there is little justification for this treatment in this location. We have designed a RCT to test the hypothesis that an oral preparation of Lactobacillus acidophilus is superior to placebo in the treatment of acute watery diarrhoea in Vietnamese children. METHODS: This RCT was designed to study the effect of treatment with L. acidophilus (4 × 109 colony-forming units/day) for 5 days for acute watery diarrhoea against a placebo in 300 children ages 9 to 60 months admitted to hospitals in Vietnam. Clinical and laboratory data plus samples will be collected on admission, daily during hospitalisation, at discharge, and at follow-up visits for a subset of participants. The primary end point will be defined as the time from the first dose of study medication to the start of the first 24-hour period without diarrhoea as assessed by the on-duty nurse. Secondary endpoints include the time to cessation of diarrhoea as recorded by parents or guardians in an hourly checklist, stool frequency over the first 3 days, treatment failure, rotavirus and norovirus viral loads, and adverse events. DISCUSSION: The existing evidence for the use of probiotics in treating acute watery diarrhoea seems to favour their use. However, the size of the effect varies across publications. An array of different probiotic organisms, doses, treatment durations, study populations, designs, settings, and aetiologies have been described. In this trial, we will investigate whether probiotics are beneficial as an adjuvant treatment for children with acute watery diarrhoea in Vietnam, with the aim of guiding clinical practice through improved regional evidence. TRIAL REGISTRATION: Current Controlled Trials ISRCTN88101063.

Phu NH, Hoang Mai NT, Nghia HD, Chau TT, Loc PP, Thai LEH, Phuong TM, Thai CQ, Man DN, Van Vinh Chau N et al. 2013. Fatal consequences of freshwater pearl diving. Lancet, 381 (9861), pp. 176. | Read more

Hoa NT, Chieu TTB, Dung SD, Long NT, Hieu TQ, Luc NT, Nhuong PT, Huong VTL, Trinh DT, Wertheim HFL et al. 2013. Streptococcus suis and porcine reproductive and respiratory syndrome, vietnam Emerging Infectious Diseases, 19 (2), pp. 331-333. | Read more

Trang NH, Nga TV, Campbell JI, Hiep NT, Farrar J, Baker S, Duy PT. 2013. The characterization of ESBL genes in Escherichia coli and Klebsiella pneumoniae causing nosocomial infections in Vietnam. J Infect Dev Ctries, 7 (12), pp. 922-928. | Show Abstract | Read more

BACKGROUND: Extended-spectrum β-lactamases (ESBLs) are enzymes capable of hydrolyzing oxyimino-β-lactams and inducing resistance to third generation cephalosporins. The genes encoding ESBLs are widespread and generally located on highly transmissible resistance plasmids. We aimed to investigate the complement of ESBL genes in E. coli and Klebsiella pneumoniae causing nosocomial infections in hospitals in Ho Chi Minh City, Vietnam. METHODOLOGY: Thirty-two non-duplicate isolates of E. coli and Klebsiella pneumoniae causing nosocomial infections, isolated between March and June 2010, were subjected to antimicrobial susceptibility testing. All isolates were PCR-amplified to detect the blaSHV, blaTEM and blaCTX-M ESBL genes and subjected to plasmid analysis. RESULTS: We found that co-resistance to multiple antimicrobials was highly prevalent, and we report the predominance of the blaCTX-M-15 and blaCTX-M-27 genes, located on highly transmissible plasmids ranging from 50 to 170 kb in size. CONCLUSIONS: Our study represents a snap shot of ESBL-producing enteric bacteria causing nosocomial infections in this setting. We suggest that antimicrobial resistance in nosocomial E. coli and Klebsiella pneumoniae is rampant in Vietnam and ESBL organisms are widespread. In view of these data and the dramatic levels of antimicrobial resistance reported in Vietnam we advocate an urgent review of antimicrobial use in the Vietnamese healthcare system.

Baker S, Duy PT, Nga TV, Dung TT, Phat VV, Chau TT, Turner AK, Farrar J, Boni MF. 2013. Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobial pressure. Elife, 2 pp. e01229. | Show Abstract | Read more

Fluoroquinolones (FQ) are the recommended antimicrobial treatment for typhoid, a severe systemic infection caused by the bacterium Salmonella enterica serovar Typhi. FQ-resistance mutations in S. Typhi have become common, hindering treatment and control efforts. Using in vitro competition experiments, we assayed the fitness of eleven isogenic S. Typhi strains with resistance mutations in the FQ target genes, gyrA and parC. In the absence of antimicrobial pressure, 6 out of 11 mutants carried a selective advantage over the antimicrobial-sensitive parent strain, indicating that FQ resistance in S. Typhi is not typically associated with fitness costs. Double-mutants exhibited higher than expected fitness as a result of synergistic epistasis, signifying that epistasis may be a critical factor in the evolution and molecular epidemiology of S. Typhi. Our findings have important implications for the management of drug-resistant S. Typhi, suggesting that FQ-resistant strains would be naturally maintained even if fluoroquinolone use were reduced. DOI:

Koirala S, Basnyat B, Arjyal A, Shilpakar O, Shrestha K, Shrestha R, Shrestha UM, Agrawal K, Koirala KD, Thapa SD et al. 2013. Gatifloxacin versus ofloxacin for the treatment of uncomplicated enteric fever in Nepal: an open-label, randomized, controlled trial. PLoS Negl Trop Dis, 7 (10), pp. e2523. | Show Abstract | Read more

BACKGROUND: Fluoroquinolones are the most commonly used group of antimicrobials for the treatment of enteric fever, but no direct comparison between two fluoroquinolones has been performed in a large randomised trial. An open-label randomized trial was conducted to investigate whether gatifloxacin is more effective than ofloxacin in the treatment of uncomplicated enteric fever caused by nalidixic acid-resistant Salmonella enterica serovars Typhi and Paratyphi A. METHODOLOGY AND PRINCIPAL FINDINGS: Adults and children clinically diagnosed with uncomplicated enteric fever were enrolled in the study to receive gatifloxacin (10 mg/kg/day) in a single dose or ofloxacin (20 mg/kg/day) in two divided doses for 7 days. Patients were followed for six months. The primary outcome was treatment failure in patients infected with nalidixic acid resistant isolates. 627 patients with a median age of 17 (IQR 9-23) years were randomised. Of the 218 patients with culture confirmed enteric fever, 170 patients were infected with nalidixic acid-resistant isolates. In the ofloxacin group, 6 out of 83 patients had treatment failure compared to 5 out of 87 in the gatifloxacin group (hazard ratio [HR] of time to failure 0.81, 95% CI 0.25 to 2.65, p = 0.73). The median time to fever clearance was 4.70 days (IQR 2.98-5.90) in the ofloxacin group versus 3.31 days (IQR 2.29-4.75) in the gatifloxacin group (HR = 1.59, 95% CI 1.16 to 2.18, p = 0.004). The results in all blood culture-confirmed patients and all randomized patients were comparable. CONCLUSION: Gatifloxacin was not superior to ofloxacin in preventing failure, but use of gatifloxacin did result in more prompt fever clearance time compared to ofloxacin. TRIAL REGISTRATION: ISRCTN 63006567 (

Shieh M, Thompson C, Phan VT, Van TT, Tediosi F, Merson L, Farrar JJ, Ha MT, Ho LV, Pham TN, Baker S. 2013. The policy of free healthcare for children under the age of 6 years in Vietnam: assessment of the uptake for children hospitalised with acute diarrhoea in Ho Chi Minh City. Trop Med Int Health, 18 (12), pp. 1444-1451. | Show Abstract | Read more

OBJECTIVE: To assess the proportion of, and reasons for, households not utilising the policy of free healthcare for children under 6 years of age (FCCU6) for hospitalisation with diarrhoea, and assess the risk of catastrophic expenditure for households that forgo FCCU6 and pay out of pocket. METHODS: Invoices detailing insurance information and charges incurred from 472 hospitalised diarrhoeal cases in one paediatric hospital in Ho Chi Minh City were retrieved. Hospital charges and the utilisation of elective services were analysed for patients utilising and not utilising FCCU6. Associations between socio-economic factors with non-utilisation of FCCU6 were evaluated. RESULTS: Overall, 29% of patients were FCCU6 non-users. The FCCU6 non-users paid a median hospital charge of $29.13 (interquartile range, IQR: $18.57-46.24), consuming no more than 1.4% of a medium-income household's annual income. Seventy per cent of low-income FCCU6 non-users utilised less-expensive elective services, whereas only 43% of medium income patients and 21% of high-income patients did (P = 0.036). Patients from larger households and those with a parent working in government were more likely to use FCCU6. CONCLUSIONS: The rate of FCCU6 non-usage in this study population was 29%. A significant proportion of those that did not use FCCU6 was from lower income households and may perceive a justifiable cost-benefit ratio when forgoing FCCU6. Although a single diarrhoeal hospitalisation is unlikely to induce a catastrophic expenditure, FCCU6 non-usage may disproportionately increase the risk of catastrophic expenditure for lower income households over multiple illnesses.

Karkey A, Thompson CN, Tran Vu Thieu N, Dongol S, Le Thi Phuong T, Voong Vinh P, Arjyal A, Martin LB, Rondini S, Farrar JJ et al. 2013. Differential epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu, Nepal: a matched case control investigation in a highly endemic enteric fever setting. PLoS Negl Trop Dis, 7 (8), pp. e2391. | Show Abstract | Read more

BACKGROUND: Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting. METHODOLOGY AND PRINCIPAL FINDINGS: A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection. CONCLUSIONS AND SIGNIFICANCE: Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.

Tan LEV, van Doorn HR, Nghia HD, Chau TT, Tu LETP, de Vries M, Canuti M, Deijs M, Jebbink MF, Baker S et al. 2013. Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections. MBio, 4 (3), pp. e00231-e00213. | Show Abstract | Read more

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.

Liang L, Juarez S, Nga TV, Dunstan S, Nakajima-Sasaki R, Davies DH, McSorley S, Baker S, Felgner PL. 2013. Immune profiling with a Salmonella Typhi antigen microarray identifies new diagnostic biomarkers of human typhoid. Sci Rep, 3 (1), pp. 1043. | Show Abstract | Read more

Current serological diagnostic assays for typhoid fever are based on detecting antibodies against Salmonella LPS or flagellum, resulting in a high false-positive rate. Here we used a protein microarray containing 2,724 Salmonella enterica serovar Typhi antigens (>63% of proteome) and identified antibodies against 16 IgG antigens and 77 IgM antigens that were differentially reactive among acute typhoid patients and healthy controls. The IgG target antigens produced a sensitivity of 97% and specificity of 80%, whereas the IgM target antigens produced 97% and 91% sensitivity and specificity, respectively. Our analyses indicated certain features such as membrane association, secretion, and protein expression were significant enriching features of the reactive antigens. About 72% of the serodiagnostic antigens were within the top 25% of the ranked antigen list using a Naïve bayes classifier. These data provide an important resource for improved diagnostics, therapeutics and vaccine development against an important human pathogen.




Dung TTN, Phat VV, Nga TVT, My PVT, Duy PT, Campbell JI, Thuy CT, Hoang NVM, Van Minh P, Le Phuc H et al. 2013. The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus Journal of Virological Methods, 187 (1), pp. 138-143. | Show Abstract | Read more

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. © 2012 Elsevier B.V.

Campbell JI, Lan NP, Qui PT, Dung LET, Farrar JJ, Baker S. 2013. A successful antimicrobial regime for Chromobacterium violaceum induced bacteremia. BMC Infect Dis, 13 (1), pp. 4. | Show Abstract | Read more

BACKGROUND: Chromobacterium violaceum is a proteobacterium found in soil and water in tropical regions. The organism rarely causes infection in humans, yet can cause a severe systemic infection by entering the bloodstream via an open wound. CASE PRESENTATION: We recently identified a case of severe bacteremia caused by Chromobacterium violaceum at the Hospital for Tropical Diseases (HTD) in Ho Chi Minh City, Vietnam. Here, we describe how rapid microbiological identification and a combination of antimicrobials was used to successfully treat this life threatening infection in a four-year-old child. CONCLUSIONS: This case shows the need for rapid diagnosis when there is the suspicion of a puncture wound contaminated with water and soil in tropical regions. We suggest that the aggressive antimicrobial combination used here is considered when this infection is suspected.

Emary K, Moore CE, Chanpheaktra N, An KP, Chheng K, Sona S, Duy PT, Nga TV, Wuthiekanun V, Amornchai P et al. 2012. Enteric fever in Cambodian children is dominated by multidrug-resistant H58 Salmonella enterica serovar Typhi with intermediate susceptibility to ciprofloxacin. Trans R Soc Trop Med Hyg, 106 (12), pp. 718-724. | Show Abstract | Read more

Infections with Salmonella enterica serovar Typhi isolates that are multidrug resistant (MDR: resistant to chloramphenicol, ampicillin, trimethoprim-sulphamethoxazole) with intermediate ciprofloxacin susceptibility are widespread in Asia but there is little information from Cambodia. We studied invasive salmonellosis in children at a paediatric hospital in Siem Reap, Cambodia. Between 2007 and 2011 Salmonella was isolated from a blood culture in 162 children. There were 151 children with enteric fever, including 148 serovar Typhi and three serovar Paratyphi A infections, and 11 children with a non-typhoidal Salmonella infection. Of the 148 serovar Typhi isolates 126 (85%) were MDR and 133 (90%) had intermediate ciprofloxacin susceptibility. Inpatient antimicrobial treatment was ceftriaxone alone or initial ceftriaxone followed by a step-down to oral ciprofloxacin or azithromycin. Complications developed in 37/128 (29%) children admitted with enteric fever and two (1.6%) died. There was one confirmed relapse. In a sample of 102 serovar Typhi strains genotyped by investigation of a subset of single nucleotide polymorphisms, 98 (96%) were the H58 haplotype, the majority of which had the common serine to phenylalanine substitution at codon 83 in the DNA gyrase. We conclude that antimicrobial-resistant enteric fever is common in Cambodian children and therapeutic options are limited.

Castonguay-Vanier J, Davong V, Bouthasavong L, Sengdetka D, Simmalavong M, Seupsavith A, Dance DA, Baker S, Le Thi Phuong T, Vongsouvath M, Newton PN. 2013. Evaluation of a simple blood culture amplification and antigen detection method for diagnosis of Salmonella enterica serovar typhi bacteremia. J Clin Microbiol, 51 (1), pp. 142-148. | Show Abstract | Read more

In most areas where typhoid is endemic, laboratory diagnosis is not possible due to the lack of appropriate facilities. We investigated whether the combination of blood culture amplification of Salmonella enterica serovar Typhi with an S. Typhi antigen rapid diagnostic test (RDT) could be an accurate and inexpensive tool for the accelerated diagnosis of patients with acute typhoid in Laos. For a panel of 23 Gram-negative reference pathogens, the Standard Diagnostics (catalog no. 15FK20; Kyonggi-do, South Korea) RDT gave positive results for S. Typhi NCTC 8385, S. Typhi NCTC 786 (Vi negative), Salmonella enterica serovar Enteritidis (ATCC 13076), and Salmonella enterica serovar Ndolo NCTC 8700 (all group D). In a prospective study of 6,456 blood culture bottles from 3,028 patients over 15 months, 392 blood culture bottles (6.1%) from 221 (7.3%) patients had Gram-negative rods (GNRs) seen in the blood culture fluid. The sensitivity, negative predictive value, specificity, and positive predictive value were 96.7%, 99.5%, 97.9%, and 87.9%, respectively, for patients with proven S. Typhi bacteremia and 91.2%, 98.4%, 98.9%, and 93.9% for patients with group D Salmonella. The median (range) number of days between diagnosis by RDT and reference assays was 1 (-1 to +2) day for those with confirmed S. Typhi. The use of antigen-based pathogen detection in blood culture fluid may be a useful, relatively rapid, inexpensive, and accurate technique for the identification of important causes of bacteremia in the tropics.

Dung TT, Phat VV, Nga TV, My PV, Duy PT, Campbell JI, Thuy CT, Hoang NV, Van Minh P, Le Phuc H et al. 2013. The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting rotavirus A and norovirus. J Virol Methods, 187 (1), pp. 138-143. | Show Abstract | Read more

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits.

Henk DA, Shahar-Golan R, Devi KR, Boyce KJ, Zhan N, Fedorova ND, Nierman WC, Hsueh PR, Yuen KY, Sieu TP et al. 2012. Clonality despite sex: the evolution of host-associated sexual neighborhoods in the pathogenic fungus Penicillium marneffei. PLoS Pathog, 8 (10), pp. e1002851. | Show Abstract | Read more

Molecular genetic approaches typically detect recombination in microbes regardless of assumed asexuality. However, genetic data have shown the AIDS-associated pathogen Penicillium marneffei to have extensive spatial genetic structure at local and regional scales, and although there has been some genetic evidence that a sexual cycle is possible, this haploid fungus is thought to be genetically, as well as morphologically, asexual in nature because of its highly clonal population structure. Here we use comparative genomics, experimental mixed-genotype infections, and population genetic data to elucidate the role of recombination in natural populations of P. marneffei. Genome wide comparisons reveal that all the genes required for meiosis are present in P. marneffei, mating type genes are arranged in a similar manner to that found in other heterothallic fungi, and there is evidence of a putatively meiosis-specific mutational process. Experiments suggest that recombination between isolates of compatible mating types may occur during mammal infection. Population genetic data from 34 isolates from bamboo rats in India, Thailand and Vietnam, and 273 isolates from humans in China, India, Thailand, and Vietnam show that recombination is most likely to occur across spatially and genetically limited distances in natural populations resulting in highly clonal population structure yet sexually reproducing populations. Predicted distributions of three different spatial genetic clusters within P. marneffei overlap with three different bamboo rat host distributions suggesting that recombination within hosts may act to maintain population barriers within P. marneffei.

Thompson CN, Phan VT, Le TP, Pham TN, Hoang LP, Ha V, Nguyen VM, Pham VM, Nguyen TV, Cao TT et al. 2013. Epidemiological features and risk factors of Salmonella gastroenteritis in children resident in Ho Chi Minh City, Vietnam. Epidemiol Infect, 141 (8), pp. 1604-1613. | Show Abstract | Read more

Non-typhoidal Salmonella are an important but poorly characterized cause of paediatric diarrhoea in developing countries. We conducted a hospital-based case-control study in children aged <5 years in Ho Chi Minh City to define the epidemiology and examine risk factors associated with Salmonella diarrhoeal infections. From 1419 diarrhoea cases and 571 controls enrolled between 2009 and 2010, 77 (5∙4%) diarrhoea cases were stool culture-positive for non-typhoidal Salmonella. Salmonella patients were more likely to be younger than controls (median age 10 and 12 months, respectively) [odds ratio (OR) 0∙97; 95% confidence interval (CI) 0∙94-0∙99], to report a recent diarrhoeal contact (8∙1% cases, 1∙8% controls; OR 5∙98, 95% CI 1∙8-20∙4) and to live in a household with >2 children (cases 20∙8%, controls 10∙2%; OR 2∙32, 95% CI 1∙2-4∙7). Our findings indicate that Salmonella are an important cause of paediatric gastroenteritis in this setting and we suggest that transmission may occur through direct human contact in the home.

Phan VTM, Ha ML, Thompson C, Nguyen VMH, Pham VM, Nguyen TV, Cao TT, Nguyen TTN, Tang CT, Hoang LP et al. 2012. Risk factors of norovirus infection and the spatiotemporal dynamics of GII.4 strain replacement in Ho Chi Minh City, Vietnam INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E101-E101. | Read more

Baker S. 2012. Solving the typhoid diagnostics conundrum INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E29-E29. | Read more

Ngoun C, Emary K, Khun PA, Moore C, Soeng S, Duy PT, Tranh NTV, Wuthiekanum V, Amonrnchai P, Kheng C et al. 2012. Enteric fever in Cambodian children is dominated by multidrug resistant H58 Salmonella enterica serovar Typhi with decreased susceptibility to ciprofloxacin INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E427-E427. | Read more

Moore C, Pan-ngum W, Wijedoru L, Ngoun C, Pastoor R, Tran N, Soeng S, Kheng C, Kumar V, Emary K et al. 2012. Evaluation of a Typhoid IgM flow assay for the diagnosis of typhoid fever in Cambodian children using a Bayesian modelling approach assuming an imperfect gold standard INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E400-E401. | Read more

Koirala KD, Thanh DP, Thapa SD, Arjyal A, Karkey A, Dongol S, Shrestha UM, Farrar JJ, Basnyat B, Baker S. 2012. Highly resistant Salmonella enterica serovar Typhi with a novel gyrA mutation raises questions about the long-term efficacy of older fluoroquinolones for treating typhoid fever. Antimicrob Agents Chemother, 56 (5), pp. 2761-2762. | Show Abstract | Read more

As a consequence of multidrug resistance, clinicians are highly dependent on fluoroquinolones for treating the serious systemic infection typhoid fever. While reduced susceptibility to fluoroquinolones, which lessens clinical efficacy, is becoming ubiquitous, comprehensive resistance is exceptional. Here we report ofloxacin treatment failure in typhoidal patient infected with a novel, highly fluoroquinolone-resistant isolate of Salmonella enterica serovar Typhi. The isolation of this organism has serious implications for the long-term efficacy of ciprofloxacin and ofloxacin for typhoid treatment.

Dongol S, Thompson CN, Clare S, Nga TV, Duy PT, Karkey A, Arjyal A, Koirala S, Khatri NS, Maskey P et al. 2012. The microbiological and clinical characteristics of invasive salmonella in gallbladders from cholecystectomy patients in kathmandu, Nepal. PLoS One, 7 (10), pp. e47342. | Show Abstract | Read more

Gallbladder carriage of invasive Salmonella is considered fundamental in sustaining typhoid fever transmission. Bile and tissue was obtained from 1,377 individuals undergoing cholecystectomy in Kathmandu to investigate the prevalence, characteristics and relevance of invasive Salmonella in the gallbladder in an endemic area. Twenty percent of bile samples contained a Gram-negative organism, with Salmonella Typhi and Salmonella Paratyphi A isolated from 24 and 22 individuals, respectively. Gallbladders that contained Salmonella were more likely to show evidence of acute inflammation with extensive neutrophil infiltrate than those without Salmonella, corresponding with higher neutrophil and lower lymphocyte counts in the blood of Salmonella positive individuals. Antimicrobial resistance in the invasive Salmonella isolates was limited, indicating that gallbladder colonization is unlikely to be driven by antimicrobial resistance. The overall role of invasive Salmonella carriage in the gallbladder is not understood; here we show that 3.5% of individuals undergoing cholecystectomy in this setting have a high concentration of antimicrobial sensitive, invasive Salmonella in their bile. We predict that such individuals will become increasingly important if current transmission mechanisms are disturbed; prospectively identifying these individuals is, therefore, paramount for rapid local and regional elimination.




Holt KE, Baker S, Weill FX, Holmes EC, Kitchen A, Yu J, Sangal V, Brown DJ, Coia JE, Kim DW et al. 2012. Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe Nature Genetics, 44 (9), pp. 1056-1059. | Show Abstract | Read more

Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery, spreading efficiently via low-dose fecal-oral transmission. Historically, S. sonnei has been predominantly responsible for dysentery in developed countries but is now emerging as a problem in the developing world, seeming to replace the more diverse Shigella flexneri in areas undergoing economic development and improvements in water quality. Classical approaches have shown that S. sonnei is genetically conserved and clonal. We report here whole-genome sequencing of 132 globally distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and that diversified into several distinct lineages with unique characteristics. Our analysis suggests that the majority of this diversification occurred in Europe and was followed by more recent establishment of local pathogen populations on other continents, predominantly due to the pandemic spread of a single, rapidly evolving, multidrug-resistant lineage. © 2012 Nature America, Inc. All rights reserved.

Vien LETM, Minh NN, Thuong TC, Khuong HD, Nga TV, Thompson C, Campbell JI, de Jong M, Farrar JJ, Schultsz C et al. 2012. The co-selection of fluoroquinolone resistance genes in the gut flora of Vietnamese children. PLoS One, 7 (8), pp. e42919. | Show Abstract | Read more

Antimicrobial consumption is one of the major contributing factors facilitating the development and maintenance of bacteria exhibiting antimicrobial resistance. Plasmid-mediated quinolone resistance (PMQR) genes, such as the qnr family, can be horizontally transferred and contribute to reduced susceptibility to fluoroquinolones. We performed an observational study, investigating the copy number of PMQR after antimicrobial therapy. We enrolled 300 children resident in Ho Chi Minh City receiving antimicrobial therapy for acute respiratory tract infections (ARIs). Rectal swabs were taken on enrollment and seven days subsequently, counts for Enterobacteriaceae were performed and qnrA, qnrB and qnrS were quantified by using real-time PCR on metagenomic stool DNA. On enrollment, we found no association between age, gender or location of the participants and the prevalence of qnrA, qnrB or qnrS. Yet, all three loci demonstrated a proportional increase in the number of samples testing positive between day 0 and day 7. Furthermore, qnrB demonstrated a significant increase in copy number between paired samples (p<0.001; Wilcoxon rank-sum), associated with non-fluoroquinolone combination antimicrobial therapy. To our knowledge, this is the first study describing an association between the use of non-fluoroquinolone antimicrobials and the increasing relative prevalence and quantity of qnr genes. Our work outlines a potential mechanism for the selection and maintenance of PMQR genes and predicts a strong effect of co-selection of these resistance determinants through the use of unrelated and potentially unnecessary antimicrobial regimes.

Holt KE, Baker S, Weill FX, Holmes EC, Kitchen A, Yu J, Sangal V, Brown DJ, Coia JE, Kim DW et al. 2012. Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe. Nat Genet, 44 (9), pp. 1056-1059. | Show Abstract | Read more

Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery(1,2), spreading efficiently via low-dose fecal-oral transmission(3,4). Historically, S. sonnei has been predominantly responsible for dysentery in developed countries but is now emerging as a problem in the developing world, seeming to replace the more diverse Shigella flexneri in areas undergoing economic development and improvements in water quality(4-6). Classical approaches have shown that S. sonnei is genetically conserved and clonal(7). We report here whole-genome sequencing of 132 globally distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and that diversified into several distinct lineages with unique characteristics. Our analysis suggests that the majority of this diversification occurred in Europe and was followed by more recent establishment of local pathogen populations on other continents, predominantly due to the pandemic spread of a single, rapidly evolving, multidrug-resistant lineage.

Seth-Smith HM, Fookes MC, Okoro CK, Baker S, Harris SR, Scott P, Pickard D, Quail MA, Churcher C, Sanders M et al. 2012. Structure, diversity, and mobility of the Salmonella pathogenicity island 7 family of integrative and conjugative elements within Enterobacteriaceae. J Bacteriol, 194 (6), pp. 1494-1504. | Show Abstract | Read more

Integrative and conjugative elements (ICEs) are self-mobile genetic elements found in the genomes of some bacteria. These elements may confer a fitness advantage upon their host bacteria through the cargo genes that they carry. Salmonella pathogenicity island 7 (SPI-7), found within some pathogenic strains of Salmonella enterica, possesses features indicative of an ICE and carries genes implicated in virulence. We aimed to identify and fully analyze ICEs related to SPI-7 within the genus Salmonella and other Enterobacteriaceae. We report the sequence of two novel SPI-7-like elements, found within strains of Salmonella bongori, which share 97% nucleotide identity over conserved regions with SPI-7 and with each other. Although SPI-7 within Salmonella enterica serovar Typhi appears to be fixed within the chromosome, we present evidence that these novel elements are capable of excision and self-mobility. Phylogenetic analyses show that these Salmonella mobile elements share an ancestor which existed approximately 3.6 to 15.8 million years ago. Additionally, we identified more distantly related ICEs, with distinct cargo regions, within other strains of Salmonella as well as within Citrobacter, Erwinia, Escherichia, Photorhabdus, and Yersinia species. In total, we report on a collection of 17 SPI-7 related ICEs within enterobacterial species, of which six are novel. Using comparative and mutational studies, we have defined a core of 27 genes essential for conjugation. We present a growing family of SPI-7-related ICEs whose mobility, abundance, and cargo variability indicate that these elements may have had a large impact on the evolution of the Enterobacteriaceae.

Nga TV, Parry CM, Le T, Lan NP, Diep TS, Campbell JI, Hoang NV, Dung LET, Wain J, Dolecek C et al. 2012. The decline of typhoid and the rise of non-typhoid salmonellae and fungal infections in a changing HIV landscape: bloodstream infection trends over 15 years in southern Vietnam. Trans R Soc Trop Med Hyg, 106 (1), pp. 26-34. | Show Abstract | Read more

The etiological spectrum of bloodstream infections is variable between industrialized and developing countries and even within a defined location over time. We investigated trends in bloodstream infections at an infectious disease hospital in Ho Chi Minh City, Vietnam, from 1994-2008. Amongst 66,111 blood cultures performed, a clinically relevant pathogen was isolated in 7645 episodes (positivity rate; 116/1000 cultures). Salmonella Typhi was the predominant pathogen until 2002; however, a considerable annual decline in the proportion of S. Typhi was observed (OR 0.6993, 95% CI [0.6885, 0.7103], p<0.0001). Conversely, there was a significant increase in the proportions of non-typhoidal Salmonella (NTS), Cryptococcus neoformans and Penicillium marneffei, concurrent with increasing HIV prevalence. These data document a substantial longitudinal shift in bloodstream infection etiology in southern Vietnam. We propose such changes are related to increasing economic prosperity and HIV prevalence, and this pattern marks a substantial change in the epidemiology of invasive salmonellosis in Southeast Asia.

Baker S. 2011. Genomic medicine has failed the poor. Nature, 478 (7369), pp. 287. | Read more

Tra My PV, Rabaa MA, Vinh H, Holmes EC, Hoang NV, Vinh NT, Phuong LET, Tham NT, Bay PV, Campbell JI et al. 2011. The emergence of rotavirus G12 and the prevalence of enteric viruses in hospitalized pediatric diarrheal patients in southern Vietnam. Am J Trop Med Hyg, 85 (4), pp. 768-775. | Show Abstract | Read more

Diarrhea is a major cause of childhood morbidity and mortality in developing countries, and the majority of infections are of viral etiology. We aimed to compare the etiological prevalence of the major enteric viruses in an urban and a rural setting in southern Vietnam. We simultaneously screened fecal specimens from 362 children in Ho Chi Minh City and Dong Thap province that were hospitalized with acute diarrhea over a 1-month-long period for four viral gastrointestinal pathogens. Rotavirus was the most common pathogen identified, but there was a differential prevalence of rotavirus and norovirus between the urban and rural locations. Furthermore, rotavirus genotyping and phylogenetic analysis again differentiated the genotypes by the sampling location. Our data show a disproportional distribution of enteric viral pathogens in urban and rural locations, and we provide evidence of continual importation of new rotavirus strains into southern Vietnam and report the emergence of rotavirus genotype G12.

Aaltonen T, Alvarez Gonzalez B, Amerio S, Amidei D, Anastassov A, Annovi A, Antos J, Apollinari G, Appel JA, Apresyan A et al. 2011. Measurements of the properties of Lambda(c)(2595), Lambda(c)(2625), Sigma(c)(2455), and Sigma(c)(2520) baryons PHYSICAL REVIEW D, 84 (1), pp. 161. | Show Abstract | Read more

We perform a search for near-threshold $\Xi_b^0$ resonances decaying to $\Xi_b^- \pi^+$ in a sample of proton-proton collision data corresponding to an integrated luminosity of 3 fb$^{-1}$ collected by the LHCb experiment. We observe one resonant state, with the following properties: \begin{eqnarray*} m(\Xi_b^{*0}) - m(\Xi_b^-) - m(\pi^+) &=& 15.727 \pm 0.068 \, (\mathrm{stat}) \pm 0.023 \, (\mathrm{syst}) \, \mathrm{MeV}/c^2, \Gamma(\Xi_b^{*0}) &=& 0.90 \pm 0.16 \, (\mathrm{stat}) \pm 0.08 \, (\mathrm{syst}) \, \mathrm{MeV} . \end{eqnarray*} This confirms the previous observation by the CMS collaboration. The state is consistent with the $J^P=3/2^+$ $\Xi_b^{*0}$ resonance expected in the quark model. This is the most precise determination of the mass and the first measurement of the natural width of this state. We have also measured the ratio \begin{align*} \frac{\sigma(pp \to \Xi_b^{*0} X){\cal{B}}(\Xi_b^{*0} \to \Xi_b^- \pi^+)}{\sigma(pp \to \Xi_b^- X)} = 0.28 \pm 0.03 \, (\mathrm{stat}) \pm 0.01 \, (\mathrm{syst}) . \end{align*}

Holt KE, Phan MD, Baker S, Duy PT, Nga TV, Nair S, Turner AK, Walsh C, Fanning S, Farrell-Ward S et al. 2011. Emergence of a globally dominant IncHI1 plasmid type associated with multiple drug resistant typhoid. PLoS Negl Trop Dis, 5 (7), pp. e1245. | Show Abstract | Read more

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains a serious global health concern. Since their emergence in the mid-1970s multi-drug resistant (MDR) S. Typhi now dominate drug sensitive equivalents in many regions. MDR in S. Typhi is almost exclusively conferred by self-transmissible IncHI1 plasmids carrying a suite of antimicrobial resistance genes. We identified over 300 single nucleotide polymorphisms (SNPs) within conserved regions of the IncHI1 plasmid, and genotyped both plasmid and chromosomal SNPs in over 450 S. Typhi dating back to 1958. Prior to 1995, a variety of IncHI1 plasmid types were detected in distinct S. Typhi haplotypes. Highly similar plasmids were detected in co-circulating S. Typhi haplotypes, indicative of plasmid transfer. In contrast, from 1995 onwards, 98% of MDR S. Typhi were plasmid sequence type 6 (PST6) and S. Typhi haplotype H58, indicating recent global spread of a dominant MDR clone. To investigate whether PST6 conferred a selective advantage compared to other IncHI1 plasmids, we used a phenotyping array to compare the impact of IncHI1 PST6 and PST1 plasmids in a common S. Typhi host. The PST6 plasmid conferred the ability to grow in high salt medium (4.7% NaCl), which we demonstrate is due to the presence in PST6 of the Tn6062 transposon encoding BetU.

Parry CM, Vinh H, Chinh NT, Wain J, Campbell JI, Hien TT, Farrar JJ, Baker S. 2011. The influence of reduced susceptibility to fluoroquinolones in Salmonella enterica serovar Typhi on the clinical response to ofloxacin therapy. PLoS Negl Trop Dis, 5 (6), pp. e1163. | Show Abstract | Read more

BACKGROUND: Infection with Salmonella enterica serovar Typhi (S. Typhi) with reduced susceptibility to fluoroquinolones has been associated with fluoroquinolone treatment failure. We studied the relationship between ofloxacin treatment response and the ofloxacin minimum inhibitory concentration (MIC) of the infecting isolate. Individual patient data from seven randomised controlled trials of antimicrobial treatment in enteric fever conducted in Vietnam in which ofloxacin was used in at least one of the treatment arms was studied. Data from 540 patients randomised to ofloxacin treatment was analysed to identify an MIC of the infecting organism associated with treatment failure. PRINCIPAL FINDINGS: The proportion of patients failing ofloxacin treatment was significantly higher in patients infected with S. Typhi isolates with an MIC≥0.25 µg/mL compared with those infections with an MIC of ≤0.125 µg/mL (p<0.001). Treatment success was 96% when the ofloxacin MIC was ≤0.125 µg/mL, 73% when the MIC was between 0.25 and 0.50 µg/mL and 53% when the MIC was 1.00 µg/mL. This was despite a longer duration of treatment at a higher dosage in patients infected with isolates with an MIC≥0.25 µg/mL compared with those infections with an MIC of ≤0.125 µg/mL. SIGNIFICANCE: There is a clear relationship between ofloxacin susceptibility and clinical outcome in ofloxacin treated patients with enteric fever. An ofloxacin MIC of ≥0.25 µg/mL, or the presence of nalidixic acid resistance, can be used to define S. Typhi infections in which the response to ofloxacin may be impaired.

Parry CM, Wijedoru L, Arjyal A, Baker S. 2011. The utility of diagnostic tests for enteric fever in endemic locations. Expert Rev Anti Infect Ther, 9 (6), pp. 711-725. | Show Abstract | Read more

Enteric fever, an infection caused by Salmonella enterica serovar Typhi and serovar Paratyphi A, is common and endemic in many areas of the Asian and African continents. In endemic areas, diagnostic tests are needed to diagnose acute cases for clinical management, to detect convalescent and chronic fecal carriage and for contact tracing. A suitable test may also allow an assessment of disease burden in a community to determine the need for vaccination programs. Each specific role may warrant a dedicated test, utilizing different samples, targets and methods to serve their respective purpose. Current diagnostic methods are poor. Blood culture is insufficiently sensitive and technically demanding, and bone marrow culture, although more sensitive, is infrequently performed. Antibody- and antigen-detection tests lend themselves to point-of-care format but remain insufficiently sensitive and specific for this role. There are concerns about the sensitivity of nucleic acid amplification tests and they have not become widely adopted. However, new approaches using genomics, proteomics, transcriptomics, in vivo-induced antigen and immunoaffinity proteomics-based technologies are being employed to identify new antigens, gene targets and metabolic products that could be used as a basis for more effective diagnostic tests. If novel tests are to be credible and widely used they require rigorous evaluation in endemic areas in studies with appropriate selection of patients, adequate sample sizes and proper attention to a gold standard reference. Here, we discuss the range of methods currently used for diagnosing enteric fever in endemic locations and we suggest new technologies which may improve enteric fever diagnostics over the coming years.

Arjyal A, Basnyat B, Koirala S, Karkey A, Dongol S, Agrawaal KK, Shakya N, Shrestha K, Sharma M, Lama S et al. 2011. Gatifloxacin versus chloramphenicol for uncomplicated enteric fever: an open-label, randomised, controlled trial. Lancet Infect Dis, 11 (6), pp. 445-454. | Show Abstract | Read more

BACKGROUND: We aimed to investigate whether gatifloxacin, a new generation and affordable fluoroquinolone, is better than chloramphenicol for the treatment of uncomplicated enteric fever in children and adults. METHODS: We did an open-label randomised superiority trial at Patan Hospital, Kathmandu, Nepal, to investigate whether gatifloxacin is more effective than chloramphenicol for treating uncomplicated enteric fever. Children and adults clinically diagnosed with enteric fever received either gatifloxacin (10 mg/kg) once a day for 7 days, or chloramphenicol (75 mg/kg per day) in four divided doses for 14 days. Patients were randomly allocated treatment (1:1) in blocks of 50, without stratification. Allocations were placed in sealed envelopes opened by the study physician once a patient was enrolled into the trial. Masking was not possible because of the different formulations and ways of giving the two drugs. The primary outcome measure was treatment failure, which consisted of at least one of the following: persistent fever at day 10, need for rescue treatment, microbiological failure, relapse until day 31, and enteric-fever-related complications. The primary outcome was assessed in all patients randomly allocated treatment and reported separately for culture-positive patients and for all patients. Secondary outcome measures were fever clearance time, late relapse, and faecal carriage. The trial is registered on, number ISRCTN 53258327. FINDINGS: 844 patients with a median age of 16 (IQR 9-22) years were enrolled in the trial and randomly allocated a treatment. 352 patients had blood-culture-confirmed enteric fever: 175 were treated with chloramphenicol and 177 with gatifloxacin. 14 patients had treatment failure in the chloramphenicol group, compared with 12 in the gatifloxacin group (hazard ratio [HR] of time to failure 0·86, 95% CI 0·40-1·86, p=0·70). The median time to fever clearance was 3·95 days (95% CI 3·68-4·68) in the chloramphenicol group and 3·90 days (3·58-4·27) in the gatifloxacin group (HR 1·06, 0·86-1·32, p=0·59). At 1 month only, three of 148 patients were stool-culture positive in the chloramphenicol group and none in the gatifloxacin group. At the end of 3 months only one person had a positive stool culture in the chloramphenicol group. There were no other positive stool cultures even at the end of 6 months. Late relapses were noted in three of 175 patients in the culture-confirmed chloramphenicol group and two of 177 in the gatifloxacin group. There were no culture-positive relapses after day 62. 99 patients (24%) experienced 168 adverse events in the chloramphenicol group and 59 (14%) experienced 73 events in the gatifloxacin group. INTERPRETATION: Although no more efficacious than chloramphenicol, gatifloxacin should be the preferred treatment for enteric fever in developing countries because of its shorter treatment duration and fewer adverse events. FUNDING: Wellcome Trust.

Wijedoru LP, Kumar V, Chanpheaktra N, Chheng K, Smits HL, Pastoor R, Nga TV, Baker S, Wuthiekanun V, Peacock SJ et al. 2012. Typhoid fever among hospitalized febrile children in Siem Reap, Cambodia. J Trop Pediatr, 58 (1), pp. 68-70. | Show Abstract | Read more

Typhoid fever was confirmed by positive blood culture in 5 (3.7%) of 134 febrile children hospitalized in Cambodia. Typhoid was suspected in an additional 25 (18.7 %) blood culture-negative children based on: a positive immunoglobulin M lateral flow assay (IgMFA) (16); a positive polymerase chain reaction (PCR) for Salmonella typhi (2); or clinical assessment (7). The specificity of the IgMFA and PCR assays requires further study.

Vien LETM, Abuoun M, Morrison V, Thomson N, Campbell JI, Woodward MJ, Van Vinh Chau N, Farrar J, Schultsz C, Baker S. 2011. Differential phenotypic and genotypic characteristics of qnrS1-harboring plasmids carried by hospital and community commensal enterobacteria. Antimicrob Agents Chemother, 55 (4), pp. 1798-1802. | Show Abstract | Read more

The qnrS1 gene induces reduced susceptibility to fluoroquinolones in enterobacteria. We investigated the structure, antimicrobial susceptibility phenotype, and antimicrobial resistance gene characteristics of qnrS1 plasmids from hospitalized patients and community controls in southern Vietnam. We found that the antimicrobial susceptibilities, resistance gene characteristics, and plasmid structures of qnrS1 plasmids from the hospital differed from those from the community. Our data imply that the characteristics of the two plasmid groups are indicative of distinct selective pressures in the differing environments.

Croucher NJ, Harris SR, Fraser C, Quail MA, Burton J, van der Linden M, McGee L, von Gottberg A, Song JH, Ko KS et al. 2011. Rapid pneumococcal evolution in response to clinical interventions. Science, 331 (6016), pp. 430-434. | Show Abstract | Read more

Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain(23F)-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.

Baker S, Holt KE, Clements AC, Karkey A, Arjyal A, Boni MF, Dongol S, Hammond N, Koirala S, Duy PT et al. 2011. Combined high-resolution genotyping and geospatial analysis reveals modes of endemic urban typhoid fever transmission. Open Biol, 1 (2), pp. 110008. | Show Abstract | Read more

Typhoid is a systemic infection caused by Salmonella Typhi and Salmonella Paratyphi A, human-restricted bacteria that are transmitted faeco-orally. Salmonella Typhi and S. Paratyphi A are clonal, and their limited genetic diversity has precluded the identification of long-term transmission networks in areas with a high disease burden. To improve our understanding of typhoid transmission we have taken a novel approach, performing a longitudinal spatial case-control study for typhoid in Nepal, combining single-nucleotide polymorphism genotyping and case localization via global positioning. We show extensive clustering of typhoid occurring independent of population size and density. For the first time, we demonstrate an extensive range of genotypes existing within typhoid clusters, and even within individual households, including some resulting from clonal expansion. Furthermore, although the data provide evidence for direct human-to-human transmission, we demonstrate an overwhelming contribution of indirect transmission, potentially via contaminated water. Consistent with this, we detected S. Typhi and S. Paratyphi A in water supplies and found that typhoid was spatially associated with public water sources and low elevation. These findings have implications for typhoid-control strategies, and our innovative approach may be applied to other diseases caused by other monophyletic or emerging pathogens.

Vinh H, Anh VT, Anh ND, Campbell JI, Hoang NV, Nga TV, Nhu NT, Minh PV, Thuy CT, Duy PT et al. 2011. A multi-center randomized trial to assess the efficacy of gatifloxacin versus ciprofloxacin for the treatment of shigellosis in Vietnamese children. PLoS Negl Trop Dis, 5 (8), pp. e1264. | Show Abstract | Read more

BACKGROUND: The bacterial genus Shigella is the leading cause of dysentery. There have been significant increases in the proportion of Shigella isolated that demonstrate resistance to nalidixic acid. While nalidixic acid is no longer considered as a therapeutic agent for shigellosis, the fluoroquinolone ciprofloxacin is the current recommendation of the World Health Organization. Resistance to nalidixic acid is a marker of reduced susceptibility to older generation fluoroquinolones, such as ciprofloxacin. We aimed to assess the efficacy of gatifloxacin versus ciprofloxacin in the treatment of uncomplicated shigellosis in children. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a randomized, open-label, controlled trial with two parallel arms at two hospitals in southern Vietnam. The study was designed as a superiority trial and children with dysentery meeting the inclusion criteria were invited to participate. Participants received either gatifloxacin (10 mg/kg/day) in a single daily dose for 3 days or ciprofloxacin (30 mg/kg/day) in two divided doses for 3 days. The primary outcome measure was treatment failure; secondary outcome measures were time to the cessation of individual symptoms. Four hundred and ninety four patients were randomized to receive either gatifloxacin (n=249) or ciprofloxacin (n=245), of which 107 had a positive Shigella stool culture. We could not demonstrate superiority of gatifloxacin and observed similar clinical failure rate in both groups (gatifloxacin; 12.0% and ciprofloxacin; 11.0%, p=0.72). The median (inter-quartile range) time from illness onset to cessation of all symptoms was 95 (66-126) hours for gatifloxacin recipients and 93 (68-120) hours for the ciprofloxacin recipients (Hazard Ratio [95%CI]=0.98 [0.82-1.17], p=0.83). CONCLUSIONS: We conclude that in Vietnam, where nalidixic acid resistant Shigellae are highly prevalent, ciprofloxacin and gatifloxacin are similarly effective for the treatment of acute shigellosis.

Karavolos MH, Bulmer DM, Spencer H, Rampioni G, Schmalen I, Baker S, Pickard D, Gray J, Fookes M, Winzer K et al. 2011. Salmonella Typhi sense host neuroendocrine stress hormones and release the toxin haemolysin E. EMBO Rep, 12 (3), pp. 252-258. | Show Abstract | Read more

Salmonella enterica serovar Typhi (S. typhi) causes typhoid fever. We show that exposure of S. typhi to neuroendocrine stress hormones results in haemolysis, which is associated with the release of haemolysin E in membrane vesicles. This effect is attributed to increased expression of the small RNA micA and RNA chaperone Hfq, with concomitant downregulation of outer membrane protein A. Deletion of micA or the two-component signal-transduction system, CpxAR, abolishes the phenotype. The hormone response is inhibited by the β-blocker propranolol. We provide mechanistic insights into the basis of neuroendocrine hormone-mediated haemolysis by S. typhi, increasing our understanding of inter-kingdom signalling.




Karavolos MH, Bulmer DM, Spencer H, Rampioni G, Schmalen I, Baker S, Pickard D, Gray J, Fookes M, Winzer K et al. 2011. Salmonella Typhi sense host neuroendocrine stress hormones and release the toxin haemolysin e EMBO Reports, 12 (3), pp. 252-258. | Show Abstract | Read more

Salmonella enterica serovar Typhi (S. typhi) causes typhoid fever. We show that exposure of S. typhi to neuroendocrine stress hormones results in haemolysis, which is associated with the release of haemolysin E in membrane vesicles. This effect is attributed to increased expression of the small RNA micA and RNA chaperone Hfq, with concomitant downregulation of outer membrane protein A. Deletion of micA or the two-component signal-transduction system, CpxAR, abolishes the phenotype. The hormone response is inhibited by the β 2-blocker propranolol. We provide mechanistic insights into the basis of neuroendocrine hormone-mediated haemolysis by S. typhi, increasing our understanding of inter-kingdom signalling. © 2011 European Molecular Biology Organization Embo.

Holt KE, Dolecek C, Chau TT, Duy PT, La TT, Hoang NV, Nga TV, Campbell JI, Manh BH, Vinh Chau NV et al. 2011. Temporal fluctuation of multidrug resistant salmonella typhi haplotypes in the mekong river delta region of Vietnam. PLoS Negl Trop Dis, 5 (1), pp. e929. | Show Abstract | Read more

BACKGROUND: typhoid fever remains a public health problem in Vietnam, with a significant burden in the Mekong River delta region. Typhoid fever is caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi), which is frequently multidrug resistant with reduced susceptibility to fluoroquinolone-based drugs, the first choice for the treatment of typhoid fever. We used a GoldenGate (Illumina) assay to type 1,500 single nucleotide polymorphisms (SNPs) and analyse the genetic variation of S. Typhi isolated from 267 typhoid fever patients in the Mekong delta region participating in a randomized trial conducted between 2004 and 2005. PRINCIPAL FINDINGS: the population of S. Typhi circulating during the study was highly clonal, with 91% of isolates belonging to a single clonal complex of the S. Typhi H58 haplogroup. The patterns of disease were consistent with the presence of an endemic haplotype H58-C and a localised outbreak of S. Typhi haplotype H58-E2 in 2004. H58-E2-associated typhoid fever cases exhibited evidence of significant geo-spatial clustering along the Sông H u branch of the Mekong River. Multidrug resistance was common in the established clone H58-C but not in the outbreak clone H58-E2, however all H58 S. Typhi were nalidixic acid resistant and carried a Ser83Phe amino acid substitution in the gyrA gene. SIGNIFICANCE: the H58 haplogroup dominates S. Typhi populations in other endemic areas, but the population described here was more homogeneous than previously examined populations, and the dominant clonal complex (H58-C, -E1, -E2) observed in this study has not been detected outside Vietnam. IncHI1 plasmid-bearing S. Typhi H58-C was endemic during the study period whilst H58-E2, which rarely carried the plasmid, was only transient, suggesting a selective advantage for the plasmid. These data add insight into the outbreak dynamics and local molecular epidemiology of S. Typhi in southern Vietnam.

Hoa NT, Chieu TT, Nghia HD, Mai NT, Anh PH, Wolbers M, Baker S, Campbell JI, Chau NV, Hien TT et al. 2011. The antimicrobial resistance patterns and associated determinants in Streptococcus suis isolated from humans in southern Vietnam, 1997-2008. BMC Infect Dis, 11 (1), pp. 6. | Show Abstract | Read more

BACKGROUND: Streptococcus suis is an emerging zoonotic pathogen and is the leading cause of bacterial meningitis in adults in Vietnam. Systematic data on the antimicrobial susceptibility profiles of S. suis strains isolated from human cases are lacking. We studied antimicrobial resistance and associated resistance determinants in S. suis isolated from patients with meningitis in southern Vietnam. METHODS: S. suis strains isolated between 1997 and 2008 were investigated for their susceptibility to six antimicrobial agents. Strains were screened for the presence and expression of tetracycline and erythromycin resistance determinants and the association of tet(M) genes with Tn916- like transposons. The localization of tetracycline resistance gene tet(L) was determined by pulse field gel electrophoresis and Southern blotting. RESULTS: We observed a significant increase in resistance to tetracycline and chloramphenicol, which was concurrent with an increase in multi-drug resistance. In tetracycline resistance strains, we identified tet(M), tet(O), tet(W) and tet(L) and confirmed their expression. All tet(M) genes were associated with a Tn916-like transposon. The co-expression of tet(L) and other tetracycline resistance gene(s) encoding for ribosomal protection protein(s) was only detected in strains with a minimum inhibitory concentration (MIC) of tetracycline of ≥ 64 mg/L. CONCLUSIONS: We demonstrated that multi-drug resistance in S. suis causing disease in humans in southern Vietnam has increased over the 11-year period studied. We report the presence and expression of tet(L) in S. suis strains and our data suggest that co-expression of multiple genes encoding distinct mechanism is required for an MIC ≥ 64 mg/L to tetracycline.

Day JN, Hoang TN, Duong AV, Hong CT, Diep PT, Campbell JI, Sieu TP, Hien TT, Bui T, Boni MF et al. 2011. Most cases of cryptococcal meningitis in HIV-uninfected patients in Vietnam are due to a distinct amplified fragment length polymorphism-defined cluster of Cryptococcus neoformans var. grubii VN1. J Clin Microbiol, 49 (2), pp. 658-664. | Show Abstract | Read more

Cryptococcal disease most commonly occurs in patients with an underlying immune deficit, most commonly HIV infection, and is due to Cryptococcus neoformans var. grubii. Occasionally disease due to this variety occurs in apparently immunocompetent patients. The relationship between strains infecting immunosuppressed and immunocompetent patients is not clear. Amplified fragment length polymorphism (AFLP) analysis was used to characterize the relationship between strains infecting HIV-infected and uninfected patients. Isolates from 51 HIV-uninfected patients and 100 HIV-infected patients with cryptococcal meningitis were compared. C. neoformans var. grubii VNI was responsible for infections in 73% of HIV-uninfected and 100% of HIV-infected patients. AFLP analysis defined two distinct clusters, VNIγ and VNIδ. The majority (84%) of isolates from HIV-uninfected patients were VNIγ, compared with only 38% of isolates from HIV-infected patients (odds ratio, 8.30; 95% confidence interval [CI], 3.04 to 26.6; P < 0.0001). In HIV-uninfected patients, underlying disease was less frequent in those with VNIγ infections. Two clusters of C. neoformans var. grubii VN1 are responsible for the majority of cases of cryptococcal meningitis in Vietnam. The distribution of these clusters differs according to the immune status of the host.

Baker S, Hanage WP, Holt KE. 2010. Navigating the future of bacterial molecular epidemiology. Curr Opin Microbiol, 13 (5), pp. 640-645. | Show Abstract | Read more

Technological advances in high-throughput genome sequencing have led to an enhanced appreciation of the genetic diversity found within populations of pathogenic bacteria. Methods based on single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) build upon the framework established by multi-locus sequence typing (MLST) and permit a detailed, targeted analysis of variation within related organisms. Robust phylogenetics, when combined with epidemiologically informative data, can be applied to study ongoing temporal and geographical fluctuations in bacterial pathogens. As genome sequencing, SNP detection and geospatial information become more accessible these methods will continue to transform the way molecular epidemiology is used to study populations of bacterial pathogens.

Parry CM, Thuy CT, Dongol S, Karkey A, Vinh H, Chinh NT, Duy PT, Thieu Nga TV, Campbell JI, Van Minh Hoang N et al. 2010. Suitable disk antimicrobial susceptibility breakpoints defining Salmonella enterica serovar Typhi isolates with reduced susceptibility to fluoroquinolones. Antimicrob Agents Chemother, 54 (12), pp. 5201-5208. | Show Abstract | Read more

Infections with Salmonella enterica serovar Typhi isolates that have reduced susceptibility to ofloxacin (MIC ≥ 0.25 μg/ml) or ciprofloxacin (MIC ≥ 0.125 μg/ml) have been associated with a delayed response or clinical failure following treatment with these antimicrobials. These isolates are not detected as resistant using current disk susceptibility breakpoints. We examined 816 isolates of S. Typhi from seven Asian countries. Screening for nalidixic acid resistance (MIC ≥ 16 μg/ml) identified isolates with an ofloxacin MIC of ≥0.25 μg/ml with a sensitivity of 97.3% (253/260) and specificity of 99.3% (552/556). For isolates with a ciprofloxacin MIC of ≥0.125 μg/ml, the sensitivity was 92.9% (248/267) and specificity was 98.4% (540/549). A zone of inhibition of ≤28 mm around a 5-μg ofloxacin disc detected strains with an ofloxacin MIC of ≥0.25 μg/ml with a sensitivity of 94.6% (246/260) and specificity of 94.2% (524/556). A zone of inhibition of ≤30 mm detected isolates with a ciprofloxacin MIC of ≥0.125 μg/ml with a sensitivity of 94.0% (251/267) and specificity of 94.2% (517/549). An ofloxacin MIC of ≥0.25 μg/ml and a ciprofloxacin MIC of ≥0.125 μg/ml detected 74.5% (341/460) of isolates with an identified quinolone resistance-inducing mutation and 81.5% (331/406) of the most common mutant (carrying a serine-to-phenylalanine mutation at codon 83 in the gyrA gene). Screening for nalidixic acid resistance or ciprofloxacin and ofloxacin disk inhibition zone are suitable for detecting S. Typhi isolates with reduced fluoroquinolone susceptibility.

Chau TT, Mai NH, Phu NH, Nghia HD, Chuong LV, Sinh DX, Duong VA, Diep PT, Campbell JI, Baker S et al. 2010. A prospective descriptive study of cryptococcal meningitis in HIV uninfected patients in Vietnam - high prevalence of Cryptococcus neoformans var grubii in the absence of underlying disease. BMC Infect Dis, 10 (1), pp. 199. | Show Abstract | Read more

BACKGROUND: Most cases of cryptococcal meningitis occur in patients with HIV infection: the course and outcome of disease in the apparently immunocompetent is much more poorly understood. We describe a cohort of HIV uninfected Vietnamese patients with cryptococcal meningitis in whom underlying disease is uncommon, and relate presenting features of patients and the characteristics of the infecting species to outcome. METHODS: A prospective descriptive study of HIV negative patients with cryptococcal meningitis based at the Hospital for Tropical Diseases, Ho Chi Minh City. All patients had comprehensive clinical assessment at baseline, were cared for by a dedicated study team, and were followed up for 2 years. Clinical presentation was compared by infecting isolate and outcome. RESULTS: 57 patients were studied. Cryptococcus neoformans var grubii molecular type VN1 caused 70% of infections; C. gattii accounted for the rest. Most patients did not have underlying disease (81%), and the rate of underlying disease did not differ by infecting species. 11 patients died while in-patients (19.3%). Independent predictors of death were age > or = 60 years and a history of convulsions (odds ratios and 95% confidence intervals 8.7 (1 - 76), and 16.1 (1.6 - 161) respectively). Residual visual impairment was common, affecting 25 of 46 survivors (54.3%). Infecting species did not influence clinical phenotype or outcome. The minimum inhibitory concentrations of flucytosine and amphotericin B were significantly higher for C. neoformans var grubii compared with C. gattii (p < 0.001 and p = 0.01 respectively). CONCLUSION: In HIV uninfected individuals in Vietnam, cryptococcal meningitis occurs predominantly in people with no clear predisposing factor and is most commonly due to C. neoformans var grubii. The rates of mortality and visual loss are high and independent of infecting species. There are detectable differences in susceptibility to commonly used antifungal drugs between species, but the clinical significance of this is not clear.

Chau TT, Campbell JI, Schultsz C, Chau NV, Diep TS, Baker S, Chinh NT, Farrar JJ, van Doorn HR. 2010. Three adult cases of Listeria monocytogenes meningitis in Vietnam. PLoS Med, 7 (7), pp. e1000306. | Read more

Nguyen NT, Ha V, Tran NV, Stabler R, Pham DT, Le TM, van Doorn HR, Cerdeño-Tárraga A, Thomson N, Campbell J et al. 2010. The sudden dominance of blaCTX-M harbouring plasmids in Shigella spp. Circulating in Southern Vietnam. PLoS Negl Trop Dis, 4 (6), pp. e702. | Show Abstract | Read more

BACKGROUND: Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges. METHODOLOGY: We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla(CTX-M) encoding plasmid. PRINCIPAL FINDINGS: We show that two different bla(CTX-M) genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla(CTX-M) gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the bla(CTX-M-24) gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids. SIGNIFICANCE: The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.

Holt KE, Baker S, Dongol S, Basnyat B, Adhikari N, Thorson S, Pulickal AS, Song Y, Parkhill J, Farrar JJ et al. 2010. High-throughput bacterial SNP typing identifies distinct clusters of Salmonella Typhi causing typhoid in Nepalese children. BMC Infect Dis, 10 (1), pp. 144. | Show Abstract | Read more

BACKGROUND: Salmonella Typhi (S. Typhi) causes typhoid fever, which remains an important public health issue in many developing countries. Kathmandu, the capital of Nepal, is an area of high incidence and the pediatric population appears to be at high risk of exposure and infection. METHODS: We recently defined the population structure of S. Typhi, using new sequencing technologies to identify nearly 2,000 single nucleotide polymorphisms (SNPs) that can be used as unequivocal phylogenetic markers. Here we have used the GoldenGate (Illumina) platform to simultaneously type 1,500 of these SNPs in 62 S. Typhi isolates causing severe typhoid in children admitted to Patan Hospital in Kathmandu. RESULTS: Eight distinct S. Typhi haplotypes were identified during the 20-month study period, with 68% of isolates belonging to a subclone of the previously defined H58 S. Typhi. This subclone was closely associated with resistance to nalidixic acid, with all isolates from this group demonstrating a resistant phenotype and harbouring the same resistance-associated SNP in GyrA (Phe83). A secondary clone, comprising 19% of isolates, was observed only during the second half of the study. CONCLUSIONS: Our data demonstrate the utility of SNP typing for monitoring bacterial populations over a defined period in a single endemic setting. We provide evidence for genotype introduction and define a nalidixic acid resistant subclone of S. Typhi, which appears to be the dominant cause of severe pediatric typhoid in Kathmandu during the study period.

Baker S, Favorov M, Dougan G. 2010. Searching for the elusive typhoid diagnostic. BMC Infect Dis, 10 (1), pp. 45. | Show Abstract | Read more

Typhoid (enteric) fever is still a common disease in many developing countries but current diagnostic tests are inadequate. Studies on pathogenesis and genomics have provided new insight into the organisms that cause enteric fever. Better understanding of the microorganisms explains, in part, why our current typhoid methodologies are limited in their diagnostic information and why developing new strategies may be a considerable challenge. Here we discuss the current position of typhoid diagnostics, highlight the need for technological improvements and suggest potential ways of advancing this area.

Karkey A, Arjyal A, Anders KL, Boni MF, Dongol S, Koirala S, My PV, Nga TV, Clements AC, Holt KE et al. 2010. The burden and characteristics of enteric fever at a healthcare facility in a densely populated area of Kathmandu. PLoS One, 5 (11), pp. e13988. | Show Abstract | Read more

Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area.

Nga TV, Karkey A, Dongol S, Thuy HN, Dunstan S, Holt K, Tu LETP, Campbell JI, Chau TT, Chau NV et al. 2010. The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens. BMC Infect Dis, 10 (1), pp. 125. | Show Abstract | Read more

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.




Nguyen NTK, Ha V, Tran NVT, Stabler R, Pham DT, Le TMV, van Doorn HR, Cerdeño-Tárraga A, Thomson N, Campbell J et al. 2010. The sudden dominance of blaCTX-M harbouring plasmids in Shigella spp. Circulating in Southern Vietnam. PLoS neglected tropical diseases, 4 (6), | Read more

Vinh H, Nhu NT, Nga TV, Duy PT, Campbell JI, Hoang NV, Boni MF, My PV, Parry C, Nga TT et al. 2009. A changing picture of shigellosis in southern Vietnam: shifting species dominance, antimicrobial susceptibility and clinical presentation. BMC Infect Dis, 9 (1), pp. 204. | Show Abstract | Read more

BACKGROUND: Shigellosis remains considerable public health problem in some developing countries. The nature of Shigellae suggests that they are highly adaptable when placed under selective pressure in a human population. This is demonstrated by variation and fluctuations in serotypes and antimicrobial resistance profile of organisms circulating in differing setting in endemic locations. Antimicrobial resistance in the genus Shigella is a constant threat, with reports of organisms in Asia being resistant to multiple antimicrobials and new generation therapies. METHODS: Here we compare microbiological, clinical and epidemiological data from patients with shigellosis over three different periods in southern Vietnam spanning 14 years. RESULTS: Our data demonstrates a shift in dominant infecting species (S. flexneri to S. sonnei) and resistance profile of the organisms circulating in southern Vietnam. We find that there was no significant variation in the syndromes associated with either S. sonnei or S. flexneri, yet the clinical features of the disease are more severe in later observations. CONCLUSIONS: Our findings show a change in clinical presentation of shigellosis in this setting, as the disease may be now more pronounced, this is concurrent with a change in antimicrobial resistance profile. These data highlight the socio-economic development of southern Vietnam and should guide future vaccine development and deployment strategies. TRIAL REGISTRATION: Current Controlled Trials ISRCTN55945881.

Le TM, Baker S, Le TP, Le TP, Cao TT, Tran TT, Nguyen VM, Campbell JI, Lam MY, Nguyen TH et al. 2009. High prevalence of plasmid-mediated quinolone resistance determinants in commensal members of the Enterobacteriaceae in Ho Chi Minh City, Vietnam. J Med Microbiol, 58 (Pt 12), pp. 1585-1592. | Show Abstract | Read more

Antimicrobial-resistant pathogenic members of the Enterobacteriaceae are a well-defined global problem. We hypothesized that one of the main reservoirs of dissemination of antimicrobial resistance genes in Vietnam is non-pathogenic intestinal flora, and sought to isolate antimicrobial-resistant organisms from hospitalized patients and non-hospitalized healthy individuals in Ho Chi Minh City. The results identified substantial faecal carriage of gentamicin-, ceftazidime- and nalidixic acid-resistant members of the Enterobacteriaceae in both hospitalized patients and non-hospitalized healthy individuals. A high prevalence of quinolone resistance determinants was identified, particularly the qnrS gene, in both community- and hospital-associated strains. Furthermore, the results demonstrated that a combination of quinolone resistance determinants can confer resistance to nalidixic acid and ciprofloxacin, even in the apparent absence of additional chromosomal resistance mutations in wild-type strains and laboratory strains with transferred plasmids. These data suggest that intestinal commensal organisms are a significant reservoir for the dissemination of plasmid-mediated quinolone resistance in Ho Chi Minh City.

Campbell JI, Lam TM, Huynh TL, To SD, Tran TT, Nguyen VM, Le TS, Nguyen VV, Parry C, Farrar JJ et al. 2009. Microbiologic characterization and antimicrobial susceptibility of Clostridium tetani isolated from wounds of patients with clinically diagnosed tetanus. Am J Trop Med Hyg, 80 (5), pp. 827-831. | Show Abstract

Clostridium tetani is the etiologic agent of the muscle-spasming disease tetanus. Despite an effective vaccine, tetanus is an ongoing problem in some developing countries. Diagnosis by bacterial culture is not done because it is generally unnecessary and the entry of route of the bacteria can be inapparent. We attempted to isolate and evaluate C. tetani from the wounds of 84 patients with tetanus. We effectively isolated C. tetani from 45 patients. All strains tested positive by polymerase chain reaction for the gene encoding tetanus neurotoxin. Antimicrobial susceptibilities were determined by disc diffusion and E-test. All C. tetani isolates were susceptible to penicillin and metronidazole but resistant to co-trimoxazole. Despite treatment with high doses of penicillin, C. tetani was isolated after 16 days of intravenous penicillin in two cases. These data show that the intravenous route for penicillin may be inadequate for clearing the infection and emphasizes wound debridement in the treatment of tetanus.

Khatri NS, Maskey P, Poudel S, Jaiswal VK, Karkey A, Koirala S, Shakya N, Agrawal K, Arjyal A, Basnyat B et al. 2009. Gallbladder carriage of Salmonella paratyphi A may be an important factor in the increasing incidence of this infection in South Asia. Ann Intern Med, 150 (8), pp. 567-568. | Read more

Vinh H, Baker S, Campbell J, Hoang NV, Loan HT, Chinh MT, Anh VT, Diep TS, Phuong LET, Schultsz C, Farrar J. 2009. Rapid emergence of third generation cephalosporin resistant Shigella spp. in Southern Vietnam. J Med Microbiol, 58 (Pt 2), pp. 281-283. | Read more

Baker S. 2009. Advances in Nucleic Acid Detection and Quantification Biochemist, 31 (1), pp. 65-66.

Le V, Le T, Cao T, Le L, Tran N, Le TP, Nguyen H, Campbell J, Baker S, Farrar J, Schultsz C. 2008. Prevalence of qnr and aac(6 ')-Ib-cr Genes in Community-Acquired Enterobacteriaceae Isolated in Healthy Volunteers in Hochiminh City INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 12 pp. E117-E118. | Read more

Baker S, Campbell JI, Stabler R, Nguyen HV, To DS, Nguyen DV, Farrar J. 2008. Fatal wound infection caused by Chromobacterium violaceum in Ho Chi Minh City, Vietnam. J Clin Microbiol, 46 (11), pp. 3853-3855. | Show Abstract | Read more

Chromobacterium violaceum is a proteobacterium found in soil and water in tropical regions which rarely causes infection in humans. Here, we report a fatal bacteremia caused by Chromobacterium violaceum in Vietnam. We describe a number of clinical, microbiological, and molecular aspects associated with this bacterial infection.

Holt KE, Parkhill J, Mazzoni CJ, Roumagnac P, Weill FX, Goodhead I, Rance R, Baker S, Maskell DJ, Wain J et al. 2008. High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi. Nat Genet, 40 (8), pp. 987-993. | Show Abstract | Read more

Isolates of Salmonella enterica serovar Typhi (Typhi), a human-restricted bacterial pathogen that causes typhoid, show limited genetic variation. We generated whole-genome sequences for 19 Typhi isolates using 454 (Roche) and Solexa (Illumina) technologies. Isolates, including the previously sequenced CT18 and Ty2 isolates, were selected to represent major nodes in the phylogenetic tree. Comparative analysis showed little evidence of purifying selection, antigenic variation or recombination between isolates. Rather, evolution in the Typhi population seems to be characterized by ongoing loss of gene function, consistent with a small effective population size. The lack of evidence for antigenic variation driven by immune selection is in contrast to strong adaptive selection for mutations conferring antibiotic resistance in Typhi. The observed patterns of genetic isolation and drift are consistent with the proposed key role of asymptomatic carriers of Typhi as the main reservoir of this pathogen, highlighting the need for identification and treatment of carriers.

Bishop A, House D, Perkins T, Baker S, Kingsley RA, Dougan G. 2008. Interaction of Salmonella enterica serovar Typhi with cultured epithelial cells: roles of surface structures in adhesion and invasion. Microbiology, 154 (Pt 7), pp. 1914-1926. | Show Abstract | Read more

In this study we investigate the ability of Salmonella enterica serovar Typhi (S. Typhi) surface structures to influence invasion and adhesion in epithelial cell assay systems. In general, S. Typhi was found to be less adherent, invasive and cytotoxic than S. enterica serovar Typhimurium (S. Typhimurium). Culture conditions had little effect on adhesion of S. Typhi to cultured cells but had a marked influence on invasion. In contrast, bacterial growth conditions did not influence S. Typhi apical invasion of polarized cells. The levels of S. Typhi, but not S. Typhimurium, invasion were increased by application of bacteria to the basolateral surface of polarized cells. Expression of virulence (Vi) capsule by S. Typhi resulted in a modest reduction in adhesion, but profoundly reduced levels of invasion of non-polarized cells. However, Vi capsule expression had no affect on invasion of the apical or basolateral surfaces of polarized cells. Mutation of the staA, tcfA or pilS genes did not affect invasion or adhesion in either the presence or the absence of Vi capsule.

Baker S, Pickard D, Whitehead S, Farrar J, Dougan G. 2008. Mobilization of the incQ plasmid R300B with a chromosomal conjugation system in Salmonella enterica serovar typhi. J Bacteriol, 190 (11), pp. 4084-4087. | Show Abstract | Read more

Salmonella pathogenicity island 7 (SPI-7) in Salmonella enterica serovar Typhi appears to be related to other genomic islands. Evidence suggests that SPI-7 is susceptible to spontaneous circularization, loss, and transposition. Here, we demonstrate that a region within SPI-7 has the ability to mobilize the small incQ plasmid R300B.

Baker S, Holt K, van de Vosse E, Roumagnac P, Whitehead S, King E, Ewels P, Keniry A, Weill FX, Lightfoot D et al. 2008. High-throughput genotyping of Salmonella enterica serovar Typhi allowing geographical assignment of haplotypes and pathotypes within an urban District of Jakarta, Indonesia. J Clin Microbiol, 46 (5), pp. 1741-1746. | Show Abstract | Read more

High-throughput epidemiological typing systems that provide phylogenetic and genotypic information are beneficial for tracking bacterial pathogens in the field. The incidence of Salmonella enterica serovar Typhi infection in Indonesia is high and is associated with atypical phenotypic traits such as expression of the j and the z66 flagellum antigens. Utilizing a high-throughput genotyping platform to investigate known nucleotide polymorphisms dispersed around the genome, we determined the haplotypes of 140 serovar Typhi isolates associated with Indonesia. We identified nine distinct serovar Typhi haplotypes circulating in Indonesia for more than 30 years, with eight of these present in a single Jakarta suburb within a 2-year period. One dominant haplotype, H59, is associated with j and z66 flagellum expression, representing a potential pathotype unique to Indonesia. Phylogenetic analysis suggests that H59 z66(+), j(+) isolates emerged relatively recently in terms of the origin of serovar Typhi and are geographically restricted. These data demonstrate the potential of high-throughput genotyping platforms for analyzing serovar Typhi populations in the field. The study also provides insight into the evolution of serovar Typhi and demonstrates the value of a molecular epidemiological technique that is exchangeable, that is internet friendly, and that has global utility.

Pickard D, Thomson NR, Baker S, Wain J, Pardo M, Goulding D, Hamlin N, Choudhary J, Threfall J, Dougan G. 2008. Molecular characterization of the Salmonella enterica serovar Typhi Vi-typing bacteriophage E1. J Bacteriol, 190 (7), pp. 2580-2587. | Show Abstract | Read more

Some bacteriophages target potentially pathogenic bacteria by exploiting surface-associated virulence factors as receptors. For example, phage have been identified that exhibit specificity for Vi capsule producing Salmonella enterica serovar Typhi. Here we have characterized the Vi-associated E1-typing bacteriophage using a number of molecular approaches. The absolute requirement for Vi capsule expression for infectivity was demonstrated using different Vi-negative S. enterica derivatives. The phage particles were shown to have an icosahedral head and a long noncontractile tail structure. The genome is 45,362 bp in length with defined capsid and tail regions that exhibit significant homology to the S. enterica transducing phage ES18. Mass spectrometry was used to confirm the presence of a number of hypothetical proteins in the Vi phage E1 particle and demonstrate that a number of phage proteins are modified posttranslationally. The genome of the Vi phage E1 is significantly related to other bacteriophages belonging to the same serovar Typhi phage-typing set, and we demonstrate a role for phage DNA modification in determining host specificity.

Karkey A, Aryjal A, Basnyat B, Baker S. 2008. Kathmandu, Nepal: still an enteric fever capital of the world. J Infect Dev Ctries, 2 (6), pp. 461-465. | Show Abstract

Kathmandu, the capital city of Nepal, has been previously coined an enteric fever capital of the world. Several studies have poignantly emphasized the significant burden of enteric fever within the local population and in travellers visiting the area. The population of Kathmandu is increasing and available figures suggest that enteric fever caused by Salmonella serovars Typhi and Paratyphi A show no significant signs of decreasing. Furthermore, our recent research demonstrates that the ratio of disease caused by these two organisms is shifting towards S. Paratyphi A. Here, we outline some of the major features of enteric fever in Kathmandu, including diagnosis, seasonal variation, transmission, and some characteristics of the infecting organisms. Our findings highlight the requirement for better understanding of the disease within the city; in turn, this will aid development of a targeted control strategy.

Baker S, Holt K, Whitehead S, Goodhead I, Perkins T, Stocker B, Hardy J, Dougan G. 2007. A linear plasmid truncation induces unidirectional flagellar phase change in H:z66 positive Salmonella Typhi. Mol Microbiol, 66 (5), pp. 1207-1218. | Show Abstract | Read more

The process by which bacteria regulate flagellar expression is known as phase variation and in Salmonella enterica this process permits the expression of one of two flagellin genes, fliC or fljB, at any one time. Salmonella Typhi (S. Typhi) is normally not capable of phase variation of flagellar antigen expression as isolates only harbour the fliC gene (H:d) and lacks an equivalent fljB locus. However, some S. Typhi isolates, exclusively from Indonesia, harbour an fljB equivalent encoded on linear plasmid, pBSSB1 that drives the expression of a novel flagellin named H:z66. H:z66+S. Typhi isolates were stimulated to change flagellar phase and genetically analysed for the mechanism of variation. The phase change was demonstrated to be unidirectional, reverting to expression from the resident chromosomal fliC gene. DNA sequencing demonstrated that pBSSB1 linear DNA was still detectable but that these derivatives had undergone deletion and were lacking fljA(z66) (encoding a flagellar repressor) and fljB(z66). The deletion end-point was found to involve one of the plasmid termini and a palindromic repeat sequence within fljB(z66), distinct to that found at the terminus of pBSSB1. These data demonstrate that, like some Streptomyces linear elements, at least one of the terminal inverted repeats of pBSSB1 is non-essential, but that a palindromic repeat sequence may be necessary for replication.

Baker S, Dougan G. 2007. The genome of Salmonella enterica serovar Typhi. Clin Infect Dis, 45 Suppl 1 (Supplement 1), pp. S29-S33. | Show Abstract | Read more

The generation of complete genome sequences provides a blueprint that facilitates the genetic characterization of pathogens and their hosts. The genome of Salmonella enterica serovar Typhi (S. Typhi) harbors ~5 million base pairs encoding some 4000 genes, of which >200 are functionally inactive. Comparison of S. Typhi isolates from around the world indicates that they are highly related (clonal) and that they emerged from a single point of origin ~30,000-50,000 years ago. Evidence suggests that, as well as undergoing gene degradation, S. Typhi has also recently acquired genes, such as those encoding the Vi antigen, by horizontal transfer events.

Baker S, Hardy J, Sanderson KE, Quail M, Goodhead I, Kingsley RA, Parkhill J, Stocker B, Dougan G. 2007. A novel linear plasmid mediates flagellar variation in Salmonella Typhi. PLoS Pathog, 3 (5), pp. e59. | Show Abstract | Read more

Unlike the majority of Salmonella enterica serovars, Salmonella Typhi (S. Typhi), the etiological agent of human typhoid, is monophasic. S. Typhi normally harbours only the phase 1 flagellin gene (fliC), which encodes the H:d antigen. However, some S. Typhi strains found in Indonesia express an additional flagellin antigen termed H:z66. Molecular analysis of H:z66+ S. Typhi revealed that the H:z66 flagellin structural gene (fljB(z66)) is encoded on a linear plasmid that we have named pBSSB1. The DNA sequence of pBSSB1 was determined to be just over 27 kbp, and was predicted to encode 33 coding sequences. To our knowledge, pBSSB1 is the first non-bacteriophage-related linear plasmid to be described in the Enterobacteriaceae.

Roumagnac P, Weill FX, Dolecek C, Baker S, Brisse S, Chinh NT, Le TA, Acosta CJ, Farrar J, Dougan G, Achtman M. 2006. Evolutionary history of Salmonella typhi. Science, 314 (5803), pp. 1301-1304. | Show Abstract | Read more

For microbial pathogens, phylogeographic differentiation seems to be relatively common. However, the neutral population structure of Salmonella enterica serovar Typhi reflects the continued existence of ubiquitous haplotypes over millennia. In contrast, clinical use of fluoroquinolones has yielded at least 15 independent gyrA mutations within a decade and stimulated clonal expansion of haplotype H58 in Asia and Africa. Yet, antibiotic-sensitive strains and haplotypes other than H58 still persist despite selection for antibiotic resistance. Neutral evolution in Typhi appears to reflect the asymptomatic carrier state, and adaptive evolution depends on the rapid transmission of phenotypic changes through acute infections.

Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, Stabler R, Thomson NR, Roberts AP, Cerdeño-Tárraga AM, Wang H et al. 2006. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet, 38 (7), pp. 779-786. | Show Abstract | Read more

We determined the complete genome sequence of Clostridium difficile strain 630, a virulent and multidrug-resistant strain. Our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. These mobile elements are putatively responsible for the acquisition by C. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. The metabolic capabilities encoded in the genome show multiple adaptations for survival and growth within the gut environment. The extreme genome variability was confirmed by whole-genome microarray analysis; it may reflect the organism's niche in the gut and should provide information on the evolution of virulence in this organism.

Anjum MF, Marooney C, Fookes M, Baker S, Dougan G, Ivens A, Woodward MJ. 2005. Identification of core and variable components of the Salmonella enterica subspecies I genome by microarray. Infect Immun, 73 (12), pp. 7894-7905. | Show Abstract | Read more

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within Salmonella enterica subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for S. enterica subspecies I strains. The majority of genes within the core component had conserved homologues in Escherichia coli K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as Shigella flexneri and Pseudomonas aeruginosa. Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons (fim, bcf, and stb) were conserved within all S. enterica strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in S. enterica serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within S.enterica subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across S. enterica subspecies I and shown how the core genome of these pathogens differs from the closely related E. coli K-12 laboratory strain.




Baker S, Sarwar Y, Aziz H, Haque A, Ali A, Dougan G, Wain J, Haque A. 2005. Detection of Vi-negative Salmonella enterica serovar Typhi in the peripheral blood of patients with typhoid fever in the Faisalabad region of Pakistan JOURNAL OF CLINICAL MICROBIOLOGY, 43 (9), pp. 4418-4425. | Read more

Baker S, Sarwar Y, Aziz H, Haque A, Ali A, Dougan G, Wain J, Haque A. 2005. Detection of Vi-negative Salmonella enterica serovar typhi in the peripheral blood of patients with typhoid fever in the Faisalabad region of Pakistan. J Clin Microbiol, 43 (9), pp. 4418-4425. | Show Abstract | Read more

The synthesis and transportation proteins of the Vi capsular polysaccharide of Salmonella enterica serovar Typhi (serovar Typhi) are encoded by the viaB operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of serovar Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative serovar Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB. Nine of the sixty strains analyzed (15%) tested negative for both tviA and tviB; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by fliC PCR for serovar Typhi; 4 of these were negative for tviA and tviB. Three of these samples tested positive for SPI-7. These results demonstrate that viaB-negative, SPI-7-positive serovar Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used.

Bishop AL, Baker S, Jenks S, Fookes M, Gaora PO, Pickard D, Anjum M, Farrar J, Hien TT, Ivens A, Dougan G. 2005. Analysis of the hypervariable region of the Salmonella enterica genome associated with tRNA(leuX). J Bacteriol, 187 (7), pp. 2469-2482. | Show Abstract | Read more

The divergence of Salmonella enterica and Escherichia coli is estimated to have occurred approximately 140 million years ago. Despite this evolutionary distance, the genomes of these two species still share extensive synteny and homology. However, there are significant differences between the two species in terms of genes putatively acquired via various horizontal transfer events. Here we report on the composition and distribution across the Salmonella genus of a chromosomal region designated SPI-10 in Salmonella enterica serovar Typhi and located adjacent to tRNA(leuX). We find that across the Salmonella genus the tRNA(leuX) region is a hypervariable hot spot for horizontal gene transfer; different isolates from the same S. enterica serovar can exhibit significant variation in this region. Many P4 phage, plasmid, and transposable element-associated genes are found adjacent to tRNA(leuX) in both Salmonella and E. coli, suggesting that these mobile genetic elements have played a major role in driving the variability of this region.

Wain J, House D, Zafar A, Baker S, Nair S, Kidgell C, Bhutta Z, Dougan G, Hasan R. 2005. Vi antigen expression in Salmonella enterica serovar Typhi clinical isolates from Pakistan. J Clin Microbiol, 43 (3), pp. 1158-1165. | Show Abstract | Read more

The accurate identification of Salmonella enterica subsp. enterica serovar Typhi variants that fail to express the capsular polysaccharide, Vi, is an important and much discussed issue for medical microbiology. We have tested a multiplex PCR method which shows the presence or absence of the genetic locus required for Vi expression. Of 2,222 Salmonella serovar Typhi clinical isolates collected from patients' blood over a 4-year period in a region of Pakistan where typhoid is endemic, 12 tested negative for Vi expression by serological agglutination. However, only 1 of these 12 was Vi negative by the multiplex PCR method. This result was confirmed by immunofluorescence, the most sensitive method for Vi characterization in Salmonella serovar Typhi. The multiplex PCR described therefore represents a simple and accurate method for surveillance for Vi-negative variants of Salmonella serovar Typhi in Pakistan. Testing of clinical isolates of Salmonella serovar Typhi, before subculture, from other regions where Vi-negative Salmonella serovar Typhi has been described should be carried out so that the impact of vaccination with purified Vi antigen on the levels of Vi-negative Salmonella serovar Typhi in bacterial populations can be assessed.

Thomson N, Baker S, Pickard D, Fookes M, Anjum M, Hamlin N, Wain J, House D, Bhutta Z, Chan K et al. 2004. The role of prophage-like elements in the diversity of Salmonella enterica serovars. J Mol Biol, 339 (2), pp. 279-300. | Show Abstract | Read more

The Salmonella enterica serovar Typhi CT18 (S.Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S.Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S.Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S.Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S.Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential "cargo" genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S.enterica.

Liu M, Gingery M, Doulatov SR, Liu Y, Hodes A, Baker S, Davis P, Simmonds M, Churcher C, Mungall K et al. 2004. Genomic and genetic analysis of Bordetella bacteriophages encoding reverse transcriptase-mediated tropism-switching cassettes. J Bacteriol, 186 (5), pp. 1503-1517. | Show Abstract | Read more

Liu et al. recently described a group of related temperate bacteriophages that infect Bordetella subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science 295: 2091-2094, 2002). Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the mtd (major tropism determinant) gene, which determines specificity for receptors on host bacteria. In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability. Forty-nine coding sequences were identified. Of these coding sequences, bbp36 contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and bpm encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these Bordetella phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes. Bordetella bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection.

Parkhill J, Sebaihia M, Preston A, Murphy LD, Thomson N, Harris DE, Holden MT, Churcher CM, Bentley SD, Mungall KL et al. 2003. Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat Genet, 35 (1), pp. 32-40. | Show Abstract | Read more

Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica are closely related Gram-negative beta-proteobacteria that colonize the respiratory tracts of mammals. B. pertussis is a strict human pathogen of recent evolutionary origin and is the primary etiologic agent of whooping cough. B. parapertussis can also cause whooping cough, and B. bronchiseptica causes chronic respiratory infections in a wide range of animals. We sequenced the genomes of B. bronchiseptica RB50 (5,338,400 bp; 5,007 predicted genes), B. parapertussis 12822 (4,773,551 bp; 4,404 genes) and B. pertussis Tohama I (4,086,186 bp; 3,816 genes). Our analysis indicates that B. parapertussis and B. pertussis are independent derivatives of B. bronchiseptica-like ancestors. During the evolution of these two host-restricted species there was large-scale gene loss and inactivation; host adaptation seems to be a consequence of loss, not gain, of function, and differences in virulence may be related to loss of regulatory or control functions.

Pickard D, Wain J, Baker S, Line A, Chohan S, Fookes M, Barron A, Gaora PO, Chabalgoity JA, Thanky N et al. 2003. Composition, acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7. J Bacteriol, 185 (17), pp. 5055-5065. | Show Abstract | Read more

Vi capsular polysaccharide production is encoded by the viaB locus, which has a limited distribution in Salmonella enterica serovars. In S. enterica serovar Typhi, viaB is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNA(pheU) sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of viaB-associated DNA in Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin isolates revealed the presence of similar SPI-7 islands. In S. enterica serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNA(pheU) site were absent. In S. enterica serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of S. enterica serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other Salmonella serovars examined, 24 contained no insertions at the equivalent tRNA(pheU) site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA(pheU)-phoN junction, which was not analyzed further. Sequence analysis of the SPI-7 region from S. enterica serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including Pseudomonas aeruginosa and Xanthomonas axonopodis pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.

Wood V, Gwilliam R, Rajandream MA, Lyne M, Lyne R, Stewart A, Sgouros J, Peat N, Hayles J, Baker S et al. 2003. The genome sequence of Schizosaccharomyces pombe (vol 415, pg 871, 2002) NATURE, 421 (6918), pp. 94-94. | Read more

Chan K, Baker S, Kim CC, Detweiler CS, Dougan G, Falkow S. 2003. Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar typhimurium DNA microarray. J Bacteriol, 185 (2), pp. 553-563. | Show Abstract | Read more

The genus Salmonella consists of over 2,200 serovars that differ in their host range and ability to cause disease despite their close genetic relatedness. The genetic factors that influence each serovar's level of host adaptation, how they evolved or were acquired, their influence on the evolution of each serovar, and the phylogenic relationships between the serovars are of great interest as they provide insight into the mechanisms behind these differences in host range and disease progression. We have used an Salmonella enterica serovar Typhimurium spotted DNA microarray to perform genomic hybridizations of various serovars and strains of both S. enterica (subspecies I and IIIa) and Salmonella bongori to gain insight into the genetic organization of the serovars. Our results are generally consistent with previously published DNA association and multilocus enzyme electrophoresis data. Our findings also reveal novel information. We observe a more distant relationship of serovar Arizona (subspecies IIIa) from the subspecies I serovars than previously measured. We also observe variability in the Arizona SPI-2 pathogenicity island, indicating that it has evolved in a manner distinct from the other serovars. In addition, we identify shared genetic features of S. enterica serovars Typhi, Paratyphi A, and Sendai that parallel their unique ability to cause enteric fever in humans. Therefore, whereas the taxonomic organization of Salmonella into serogroups provides a good first approximation of genetic relatedness, we show that it does not account for genomic changes that contribute to a serovar's degree of host adaptation.

Hall N, Pain A, Berriman M, Churcher C, Harris B, Harris D, Mungall K, Bowman S, Atkin R, Baker S et al. 2002. Sequence of Plasmodium falciparum chromosomes 1, 3-9 and 13. Nature, 419 (6906), pp. 527-531. | Show Abstract | Read more

Since the sequencing of the first two chromosomes of the malaria parasite, Plasmodium falciparum, there has been a concerted effort to sequence and assemble the entire genome of this organism. Here we report the sequence of chromosomes 1, 3-9 and 13 of P. falciparum clone 3D7--these chromosomes account for approximately 55% of the total genome. We describe the methods used to map, sequence and annotate these chromosomes. By comparing our assemblies with the optical map, we indicate the completeness of the resulting sequence. During annotation, we assign Gene Ontology terms to the predicted gene products, and observe clustering of some malaria-specific terms to specific chromosomes. We identify a highly conserved sequence element found in the intergenic region of internal var genes that is not associated with their telomeric counterparts.




Hall N, Pain A, Berriman M, Churcher C, Harris B, Harris D, Mungall K, Bowman S, Atkin R, Baker S et al. 2002. Sequence of Plasmodium falciparum chromosomes 1, 3–9 and 13 Nature, 419 (6906), pp. 527-531. | Read more

Wood V, Gwilliam R, Rajandream MA, Lyne M, Lyne R, Stewart A, Sgouros J, Peat N, Hayles J, Baker S et al. 2002. The genome sequence of Schizosaccharomyces pombe. Nature, 415 (6874), pp. 871-880. | Show Abstract | Read more

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.

Parkhill J, Dougan G, James KD, Thomson NR, Pickard D, Wain J, Churcher C, Mungall KL, Bentley SD, Holden MT et al. 2001. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature, 413 (6858), pp. 848-852. | Show Abstract | Read more

Salmonella enterica serovar Typhi (S. typhi) is the aetiological agent of typhoid fever, a serious invasive bacterial disease of humans with an annual global burden of approximately 16 million cases, leading to 600,000 fatalities. Many S. enterica serovars actively invade the mucosal surface of the intestine but are normally contained in healthy individuals by the local immune defence mechanisms. However, S. typhi has evolved the ability to spread to the deeper tissues of humans, including liver, spleen and bone marrow. Here we have sequenced the 4,809,037-base pair (bp) genome of a S. typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands. Notably, the genome sequence identifies over two hundred pseudogenes, several corresponding to genes that are known to contribute to virulence in Salmonella typhimurium. This genetic degradation may contribute to the human-restricted host range for S. typhi. CT18 harbours a 218,150-bp multiple-drug-resistance incH1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis.

Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MT, Prentice MB, Sebaihia M, James KD, Churcher C, Mungall KL et al. 2001. Genome sequence of Yersinia pestis, the causative agent of plague. Nature, 413 (6855), pp. 523-527. | Show Abstract | Read more

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.

Townsend SM, Kramer NE, Edwards R, Baker S, Hamlin N, Simmonds M, Stevens K, Maloy S, Parkhill J, Dougan G, Bäumler AJ. 2001. Salmonella enterica serovar Typhi possesses a unique repertoire of fimbrial gene sequences. Infect Immun, 69 (5), pp. 2894-2901. | Show Abstract | Read more

Salmonella enterica serotype Typhi differs from nontyphoidal Salmonella serotypes by its strict host adaptation to humans and higher primates. Since fimbriae have been implicated in host adaptation, we investigated whether the serotype Typhi genome contains fimbrial operons which are unique to this pathogen or restricted to typhoidal Salmonella serotypes. This study established for the first time the total number of fimbrial operons present in an individual Salmonella serotype. The serotype Typhi CT18 genome, which has been sequenced by the Typhi Sequencing Group at the Sanger Centre, contained a type IV fimbrial operon, an orthologue of the agf operon, and 12 putative fimbrial operons of the chaperone-usher assembly class. In addition to sef, fim, saf, and tcf, which had been described previously in serotype Typhi, we identified eight new putative chaperone-usher-dependent fimbrial operons, which were termed bcf, sta, stb, ste, std, stc, stg, and sth. Hybridization analysis performed with 16 strains of Salmonella reference collection C and 22 strains of Salmonella reference collection B showed that all eight putative fimbrial operons of serotype Typhi were also present in a number of nontyphoidal Salmonella serotypes. Thus, a simple correlation between host range and the presence of a single fimbrial operon seems at present unlikely. However, the serotype Typhi genome differed from that of all other Salmonella serotypes investigated in that it contained a unique combination of putative fimbrial operons.

Prentice MB, James KD, Parkhill J, Baker SG, Stevens K, Simmonds MN, Mungall KL, Churcher C, Oyston PC, Titball RW et al. 2001. Yersinia pestis pFra shows biovar-specific differences and recent common ancestry with a Salmonella enterica serovar Typhi plasmid. J Bacteriol, 183 (8), pp. 2586-2594. | Show Abstract | Read more

Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y. pestis-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different Y. pestis strains which distinguish Y. pestis bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between Y. pestis and the exclusively human pathogen Salmonella enterica serovar Typhi.


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