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Abstract Challenges in classifying recurrent Plasmodium vivax infections constrain surveillance of antimalarial efficacy and transmission. Recurrent infections may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or reinfection. Molecular inference of familial relatedness (identity-by-descent or IBD) can help resolve the probable origin of recurrences. As whole genome sequencing of P. vivax remains challenging, targeted genotyping methods are needed for scalability. We describe a P. vivax marker discovery framework to identify and select panels of microhaplotypes (multi-allelic markers within small, amplifiable segments of the genome) that can accurately capture IBD. We evaluate panels of 50–250 microhaplotypes discovered in a global set of 615 P. vivax genomes. A candidate global 100-microhaplotype panel exhibits high marker diversity in the Asia-Pacific, Latin America and horn of Africa (median H E = 0.70–0.81) and identifies 89% of the polyclonal infections detected with genome-wide datasets. Data simulations reveal lower error in estimating pairwise IBD using microhaplotypes relative to traditional biallelic SNP barcodes. The candidate global panel also exhibits high accuracy in predicting geographic origin and captures local infection outbreak and bottlenecking events. Our framework is open-source enabling customised microhaplotype discovery and selection, with potential for porting to other species or data resources.

The evolution of influenza viruses is fundamentally shaped by within-host processes. However, the within-host evolutionary dynamics of influenza viruses remain incompletely understood, in part because most studies have focused on infections in healthy adults based on single timepoint data. Here, we analyzed the within-host evolution of 82 longitudinally sampled individuals, mostly young children, infected with A/H1N1pdm09 or A/H3N2 viruses between 2007 and 2009. For A/H1N1pdm09 infections during the 2009 pandemic, nonsynonymous minority variants were more prevalent than synonymous ones. For A/H3N2 viruses in young children, early infection was dominated by purifying selection. As these infections progressed, nonsynonymous variants typically increased in frequency even when within-host virus titers decreased. Unlike the short-lived infections of adults where de novo within-host variants are rare, longer infections in young children allow for the maintenance of virus diversity via mutation-selection balance creating potentially important opportunities for within-host virus evolution.

The dominant viral etiologies responsible for acute respiratory infections (ARIs) are poorly understood, particularly among hospitalized children in resource-limited tropical countries where morbidity and mortality caused by ARIs are highest. Improved etiological insight is needed to improve clinical management and prevention.We conducted a three-year prospective descriptive study of severe respiratory illness among children from 2 months to 13 years of age within the largest referral hospital for infectious diseases in southern Vietnam.Molecular detection for 15 viral species and subtypes was performed on three types of respiratory specimens (nose, throat swabs and nasopharyngeal aspirates) using a multiplex RT-PCR kit (Seeplex™ RV detection, Seegene) and additional monoplex real-time RT-PCRs.A total of 309 children were enrolled from November 2004 to January 2008. Viruses were identified in 72% (222/309) of cases, including respiratory syncytial virus (24%), influenza virus A and B (17%), human bocavirus (16%), enterovirus (9%), human coronavirus (8%), human metapneumovirus (7%), parainfluenza virus 1-3 (6%), adenovirus (5%), and human rhinovirus A (4%). Co-infections with multiple viruses were detected in 20% (62/309) of patients. When combined, diagnostic yields in nose and throat swabs were similar to nasopharyngeal aspirates.Similar to other parts in the world, RSV and influenza were the predominant viral pathogens detected in Vietnamese hospitalized children. Combined nasal and throat swabs are the specimens of choice for sensitive molecular detection of a broad panel of viral agents. Further research is required to better understand the clinical significance of single versus multiple viral coinfections and to address the role of bacterial (co-)infections involved in severe respiratory illness.

Streptococcus suis is a pathogen of major economic significance to the swine industry and is increasingly recognized as an emerging zoonotic agent in Asia. In Vietnam, S. suis is the leading cause of bacterial meningitis in adult humans. Zoonotic transmission is most frequently associated with serotype 2 strains and occupational exposure to pigs or consumption of infected pork. To gain insight into the role of pigs for human consumption as a reservoir for zoonotic infection in southern Vietnam, we determined the prevalence and diversity of S. suis carriage in healthy slaughterhouse pigs. Nasopharyngeal tonsils were sampled from pigs at slaughterhouses serving six provinces in southern Vietnam and Ho Chi Minh City area from September 2006 to November 2007. Samples were screened by bacterial culture. Isolates of S. suis were serotyped and characterized by multi locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Antibiotic susceptibility profiles and associated genetic resistance determinants, and the presence of putative virulence factors were determined. 41% (222/542) of pigs carried S. suis of one or multiple serotypes. 8% (45/542) carried S. suis serotype 2 which was the most common serotype found (45/317 strains, 14%). 80% of serotype 2 strains belonged to the MLST clonal complex 1,which was previously associated with meningitis cases in Vietnam and outbreaks of severe disease in China in 1998 and 2005. These strains clustered with representative strains isolated from patients with meningitis in PFGE analysis, and showed similar antimicrobial resistance and virulence factor profiles. Slaughterhouse pigs are a major reservoir of S. suis serotype 2 capable of causing human infection in southern Vietnam. Strict hygiene at processing facilities, and health education programs addressing food safety and proper handling of pork should be encouraged.

Optimising the fluid resuscitation of patients with severe malaria is a simple and potentially cost-effective intervention. Current WHO guidelines recommend central venous pressure (CVP) guided, crystalloid based, resuscitation in adults.Prospectively collected haemodynamic data from intervention trials in Vietnamese adults with severe malaria were analysed retrospectively to assess the responses to fluid resuscitation.43 patients were studied of whom 24 received a fluid load. The fluid load resulted in an increase in cardiac index (mean increase: 0.75 L/min/m(2) (95% Confidence interval (CI): 0.41 to 1.1)), but no significant change in acid-base status post resuscitation (mean increase base deficit 0.6 mmol/L (95% CI: -0.1 to 1.3). The CVP and PAoP (pulmonary artery occlusion pressure) were highly inter-correlated (r(s) = 0.7, p

Dexamethasone has been shown to reduce mortality in patients with tuberculous meningitis but the long-term outcome of the disease is unknown.Vietnamese adults and adolescents with tuberculous meningitis recruited to a randomised, double-blind, placebo-controlled trial of adjunctive dexamethasone were followed-up at five years, to determine the effect of dexamethasone on long-term survival and neurological disability.545 patients were randomised to receive either dexamethasone (274 patients) or placebo (271 patients). 50 patients (9.2%) were lost to follow-up at five years. In all patients two-year survival, probabilities tended to be higher in the dexamethasone arm (0.63 versus 0.55; p = 0.07) but five-year survival rates were similar (0.54 versus 0.51, p = 0.51) in both groups. In patients with grade 1 TBM, but not with grade 2 or grade 3 TBM, the benefit of dexamethasone treatment tended to persist over time (five-year survival probabilities 0.69 versus 0.55, p = 0.07) but there was no conclusive evidence of treatment effect heterogeneity by TBM grade (p = 0.36). The dexamethasone group had a similar proportion of severely disabled patients among survivors at five years as the placebo group (17/128, 13.2% vs. 17/116, 14.7%) and there was no significant association between dexamethasone treatment and disability status at five years (p = 0.32).Adjunctive dexamethasone appears to improve the probability of survival in patients with TBM, until at least two years of follow-up. We could not demonstrate a five-year survival benefit of dexamethasone treatment which may be confined to patients with grade 1 TBM.ClinicalTrials.gov NCT01317654.

Acute encephalitis is an important and severe disease in children in Vietnam. However, little is known about the etiology while such knowledge is essential for optimal prevention and treatment. To identify viral causes of encephalitis, in 2004 we conducted a one-year descriptive study at Children's Hospital Number One, a referral hospital for children in southern Vietnam including Ho Chi Minh City.Children less than 16 years of age presenting with acute encephalitis of presumed viral etiology were enrolled. Diagnostic efforts included viral culture, serology and real time (RT)-PCRs. A confirmed or probable viral causative agent was established in 41% of 194 enrolled patients. The most commonly diagnosed causative agent was Japanese encephalitis virus (n = 50, 26%), followed by enteroviruses (n = 18, 9.3%), dengue virus (n = 9, 4.6%), herpes simplex virus (n = 1), cytomegalovirus (n = 1) and influenza A virus (n = 1). Fifty-seven (29%) children died acutely. Fatal outcome was independently associated with patient age and Glasgow Coma Scale (GCS) on admission.Acute encephalitis in children in southern Vietnam is associated with high mortality. Although the etiology remains unknown in a majority of the patients, the result from the present study may be useful for future design of treatment and prevention strategies of the disease. The recognition of GCS and age as predictive factors may be helpful for clinicians in managing the patient.

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC(-)ssaV(-)) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.Subjects were randomly assigned to receive either a nominal dose of 5x10(9) CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18-5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12-36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI] = 1.23 [0.550-2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC(-)ssaV(-)) ZH9 was detected in 51 (51%; 95% CI, 41-61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92-99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7-29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88-100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.Controlled-trials.com ISRCTN91111837.

A better description of the extent and structure of genetic diversity in dengue virus (DENV) in endemic settings is central to its eventual control. To this end we determined the complete coding region sequence of 187 DENV-2 genomes and 68 E genes from viruses sampled from Vietnamese patients between 1995 and 2009. Strikingly, an episode of genotype replacement was observed, with Asian 1 lineage viruses entirely displacing the previously dominant Asian/American lineage viruses. This genotype replacement event also seems to have occurred within DENV-2 in Thailand and Cambodia, suggestive of a major difference in viral fitness. To determine the cause of this major evolutionary event we compared both the infectivity of the Asian 1 and Asian/American genotypes in mosquitoes and their viraemia levels in humans. Although there was little difference in infectivity in mosquitoes, we observed significantly higher plasma viraemia levels in paediatric patients infected with Asian 1 lineage viruses relative to Asian/American viruses, a phenotype that is predicted to result in a higher probability of human-to-mosquito transmission. These results provide a mechanistic basis to a marked change in the genetic structure of DENV-2 and more broadly underscore that an understanding of DENV evolutionary dynamics can inform the development of vaccines and anti-viral drugs.

Understanding the host genetic susceptibility to typhoid fever may provide a better understanding of pathogenesis and help in the development of new therapeutics and vaccines. Here we determine the genetic variation within the human TLR4 gene encoding the principal receptor for bacterial endotoxin recognition in typhoid fever patients. It is possible that genetic variants of TLR4 could detrimentally affect the innate immune response against S. typhi infection. Mutation detection and genotyping of TLR4 was performed on DNA from 414 Vietnamese typhoid fever patients and 372 population controls. dHPLC detected a total of 10 polymorphisms within the upstream and exonic regions of TLR4, of which 7 are novel. Two SNPs, T4025A and C4215G, were more frequent in typhoid cases than in controls however due to their low allele frequencies they showed borderline significance (T4025A: OR 1.9, 95%CI 0.9-4.3, P 0.07 and C4215G: OR 6.7, 95%CI 0.8-307, P 0.04). Six missense mutations were identified, with 5/6 positioned in the ectoplasmic domain. Four missense mutations and one promoter SNP (A-271G) were only present in typhoid cases, albeit at low allele frequencies. Here we determined the extent of genetic variation within TLR4 in a Vietnamese population and suggest that TLR4 may be involved in defense against typhoid fever in this population.

Drug resistant typhoid fever is a major clinical problem globally. Many of the first line antibiotics, including the older generation fluoroquinolones, ciprofloxacin and ofloxacin, are failing.We performed a randomised controlled trial to compare the efficacy and safety of gatifloxacin (10 mg/kg/day) versus azithromycin (20 mg/kg/day) as a once daily oral dose for 7 days for the treatment of uncomplicated typhoid fever in children and adults in Vietnam.An open-label multi-centre randomised trial with pre-specified per protocol analysis and intention to treat analysis was conducted. The primary outcome was fever clearance time, the secondary outcome was overall treatment failure (clinical or microbiological failure, development of typhoid fever-related complications, relapse or faecal carriage of S. typhi).We enrolled 358 children and adults with suspected typhoid fever. There was no death in the study. 287 patients had blood culture confirmed typhoid fever, 145 patients received gatifloxacin and 142 patients received azithromycin. The median FCT was 106 hours in both treatment arms (95% Confidence Interval [CI]; 94-118 hours for gatifloxacin versus 88-112 hours for azithromycin), (logrank test p = 0.984, HR [95% CI] = 1.0 [0.80-1.26]). Overall treatment failure occurred in 13/145 (9%) patients in the gatifloxacin group and 13/140 (9.3%) patients in the azithromycin group, (logrank test p = 0.854, HR [95% CI] = 0.93 [0.43-2.0]). 96% (254/263) of the Salmonella enterica serovar Typhi isolates were resistant to nalidixic acid and 58% (153/263) were multidrug resistant.Both antibiotics showed an excellent efficacy and safety profile. Both gatifloxacin and azithromycin can be recommended for the treatment of typhoid fever particularly in regions with high rates of multidrug and nalidixic acid resistance. The cost of a 7-day treatment course of gatifloxacin is approximately one third of the cost of azithromycin in Vietnam.Controlled-Trials.com ISRCTN67946944.

Infections with community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are emerging worldwide. We investigated an outbreak of severe CA-MRSA infections in children following out-patient vaccination.We carried out a field investigation after adverse events following immunization (AEFI) were reported. We reviewed the clinical data from all cases. S. aureus recovered from skin infections and from nasal and throat swabs were analyzed by pulse-field gel electrophoresis, multi locus sequence typing, PCR and microarray. In May 2006, nine children presented with AEFI, ranging from fatal toxic shock syndrome, necrotizing soft tissue infection, purulent abscesses, to fever with rash. All had received a vaccination injection in different health centres in one District of Ho Chi Minh City. Eight children had been vaccinated by the same health care worker (HCW). Deficiencies in vaccine quality, storage practices, or preparation and delivery were not found. Infection control practices were insufficient. CA-MRSA was cultured in four children and from nasal and throat swabs from the HCW. Strains from children and HCW were indistinguishable. All carried the Panton-Valentine leukocidine (PVL), the staphylococcal enterotoxin B gene, the gene complex for staphylococcal-cassette-chromosome mec type V, and were sequence type 59. Strain HCM3A is epidemiologically unrelated to a strain of ST59 prevalent in the USA, although they belong to the same lineage.We describe an outbreak of infections with CA-MRSA in children, transmitted by an asymptomatic colonized HCW during immunization injection. Consistent adherence to injection practice guidelines is needed to prevent CA-MRSA transmission in both in- and outpatient settings.

Background: Acute respiratory infections (ARI) are among the leading causes of hospitalization in children ≤5 years old. Rapid diagnostics of viral pathogens is essential to avoid unnecessary antibiotic treatment, thereby slowing down antibiotic-resistance. We evaluated the diagnostic performance of the Luminex xTAG Respiratory Viral Panel FAST v2 against viral specific PCR as reference assays for ARI in Vietnam., Methods: Four hundred and forty two nose and throat swabs were collected in viral transport medium, and were tested with Luminex xTAG Respiratory Viral Panel FAST v2. Multiplex RT-PCR and single RT-PCR were used as references.    Results: Overall, viral pathogens were detected in a total count of 270/294 (91.8%, 95% CI 88.1-94.7) by the Luminex among reference assays, whilst 112/6336 (1.8%, 95% CI, 1.4-2.1) of pathogens were detected by the Luminex, but not by reference assays. Frequency of pathogens detected by Luminex and reference assays was 379 and 292, respectively. The diagnostic yield was 66.7% (295/442, 95%CI 62.1-71.1%) for the Luminex assay and 54.1% (239/442, 95% CI, 49.3-58.8%) for reference assays. The Luminex kit had higher yields for all viruses except influenza B virus, respiratory syncytial virus, and human bocavirus. High agreements between both methods [mean (range): 0.91 (0.83-1.00)] were found for 10/15 viral agents., Conclusions: The Luminex assay is a high throughput multiplex platform for rapid detection of common viral pathogens causing ARI. Although the current high cost may prevent Luminex assays from being widely used, especially in limited resource settings where ARI are felt most, its introduction in clinical diagnostics may help reduce unnecessary use of antibiotic prescription., Competing Interests: Competing interests: No competing interests were disclosed.

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., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

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.

Background: Tuberculous meningitis occurs more commonly in human immunodeficiency virus (HIV)-infected individuals than in HIV-uninfected individuals, but whether HIV infection alters the presentation and outcome of tuberculous meningitis is unknown., Methods: We performed a prospective comparison of the presenting clinical features and response to treatment in 528 adults treated consecutively for tuberculous meningitis (96 were infected with HIV and 432 were uninfected with HIV) in 2 tertiary-care referral hospitals in Ho Chi Minh City, Vietnam. Logistic regression was used to model variables associated independently with HIV infection, 9-month survival, and the likelihood of having a relapse or an adverse drug event. Kaplan-Meier estimates were used to compare survival rates and times to fever clearance, coma clearance, relapse, and adverse events., Results: HIV infection did not alter the neurological presentation of tuberculous meningitis, although additional extrapulmonary tuberculosis was more likely to occur in HIV-infected patients. The 9-month survival rate was significantly decreased in HIV-infected patients (relative risk of death from any cause, 2.91 [95% confidence interval, 2.14-3.96]; P < .001), although the times to fever clearance and coma clearance and the number or timing of relapses or adverse drug events were not significantly different between the groups., Conclusions: HIV infection does not alter the neurological features of tuberculous meningitis but significantly reduces the survival rate.

The pathogenesis of tuberculous meningitis remains unclear, and there are few data describing the kinetics of the immune response during the course of its treatment. We measured concentrations of pro- and anti-inflammatory cytokines in serial blood and cerebrospinal fluid (CSF) samples from 21 adults who were being treated for tuberculous meningitis. CSF concentrations of soluble tumor necrosis factor-alpha receptors and of matrix metalloprotein-9 and its tissue inhibitor were also measured, and blood-brain barrier permeability was assessed by the albumin and IgG partition indices. CSF concentrations of lactate, interleukin-8, and interferon-gamma were high before treatment and then decreased rapidly with antituberculosis chemotherapy. However, significant immune activation and blood-brain barrier dysfunction were still apparent after 60 days of treatment. Death was associated with high initial CSF concentrations of lactate, low numbers of white blood cells, in particular neutrophils, and low CSF glucose levels.