Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.

Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam.

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.

Hepatitis E in southern Vietnam: Seroepidemiology in humans and molecular epidemiology in pigs.

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.

A spatial and temporal analysis of paediatric central nervous system infections from 2005 to 2015 in Ho Chi Minh City, Vietnam.

Central nervous system infections (CNSI) are a leading cause of death and long-term disability in children. Using ICD-10 data from 2005 to 2015 from three central hospitals in Ho Chi Minh City (HCMC), Vietnam, we exploited generalized additive mixed models (GAMM) to examine the spatial-temporal distribution and spatial and climatic risk factors of paediatric CNSI, excluding tuberculous meningitis, in this setting. From 2005 to 2015, there were 9469 cases of paediatric CNSI; 33% were ⩽1 year old at admission and were mainly diagnosed with presumed bacterial CNSI (BI) (79%), the remainder were >1 year old and mainly diagnosed with presumed non-bacterial CNSI (non-BI) (59%). The urban districts of HCMC in proximity to the hospitals as well as some outer districts had the highest incidences of BI and non-BI; BI incidence was higher in the dry season. Monthly BI incidence exhibited a significant decreasing trend over the study. Both BI and non-BI were significantly associated with lags in monthly average temperature, rainfall, and river water level. Our findings add new insights into this important group of infections in Vietnam, and highlight where resources for the prevention and control of paediatric CNSI should be allocated.

A polymorphism in human MR1 is associated with mRNA expression and susceptibility to tuberculosis.

The MR1 antigen-presenting system is conserved among mammals and enables T cells to recognize small molecules produced by bacterial pathogens, including Mycobacterium tuberculosis (M.tb). However, it is not known whether MR1-mediated antigen presentation is important for protective immunity against mycobacterial disease. We hypothesized that genetic control of MR1 expression correlates with clinical outcomes of tuberculosis infection. We performed an MR1 candidate gene association study and identified an intronic single-nucleotide polymorphism (rs1052632) that was significantly associated with susceptibility to tuberculosis in a discovery and validation cohort of Vietnamese adults with tuberculosis. Stratification by site of disease revealed that rs1052632 genotype GG was strongly associated with the development of meningeal tuberculosis (odds ratio=2.99; 95% confidence interval (CI) 1.64-5.43; P=0.00006). Among patients with meningeal disease, absence of the G allele was associated with an increased risk of death (hazard ratio=3.86; 95% CI 1.49-9.98; P=0.005). Variant annotation tools using public databases indicate that rs1052632 is strongly associated with MR1 gene expression in lymphoblastoid cells (P=0.004) and is located within a transcriptional enhancer in epithelial keratinocytes. These data support a role for MR1 in the pathogenesis of human tuberculosis by revealing that rs1052632 is associated with MR1 gene expression and susceptibility to tuberculosis in Vietnam.

MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage.

Macrophage receptor with collagenous structure (MARCO) has an important role in the phagocytosis of Mycobacterium tuberculosis (M. tuberculosis). We hypothesized that MARCO polymorphisms are associated with phagocytosis, tuberculosis (TB) disease susceptibility and presentation, and infecting lineage. We used a human cellular model to examine how MARCO genotype mediates the immune response; a case-control study to investigate tuberculosis host genetic susceptibility; and a host-pathogen genetic analysis to study host-pathogen interactions. Two MARCO heterozygous (AG) genotypes (single-nucleotide polymorphisms rs2278589 and rs6751745) were associated with impaired phagocytosis of M. tuberculosis trehalose 6,6'-dimycolate-cord factor and ß-glucan-coated beads in macrophages. The heterozygous genotypes of rs2278589 and rs6751745 were also associated with increased risk of pulmonary TB (PTB; rs2278589, P=0.001, odds ratio (OR)=1.6; rs6751745, P=0.009, OR=1.4), and with severe chest X-ray abnormalities (P=0.007, OR=1.6). These two genotypes were also associated with the Beijing lineage (rs2278589, P=0.001, OR=1.7; rs6751745, P=0.01, OR=1.5). Together, these results suggest that MARCO polymorphisms may regulate phagocytosis of M. tuberculosis and susceptibility and severity of PTB. They also suggest MARCO genotype and Beijing strains may interact to increase the risk of PTB.

Highly Pathogenic Avian Influenza Virus A/H5N1 Infection in Vaccinated Meat Duck Flocks in the Mekong Delta of Vietnam.

We investigated episodes of suspected highly pathogenic avian influenza (HPAI)-like illness among 12 meat duck flocks in two districts in Tien Giang province (Mekong Delta, Vietnam) in November 2013. In total, duck samples from 8 of 12 farms tested positive for HPAI virus subtype A/haemagglutinin 5 and neuraminidase 1 (H5N1) by real-time RT-PCR. Sequencing results confirmed clade of 2.3.2.1.c as the cause of the outbreaks. Most (7/8) laboratory-confirmed positive flocks had been vaccinated with inactivated HPAI H5N1 clade 2.3.4 vaccines <6 days prior to onset of clinical signs. A review of vaccination data in relation to estimated production in the area suggested that vaccination efforts were biased towards larger flocks and that vaccination coverage was low [21.2% ducks vaccinated with two shots (range by district 7.4-34.9%)]. The low-coverage data, the experimental evidence of lack of cross-protection conferred by the currently used vaccines based on clade 2.3.4 together with the short lifespan of meat duck flocks (60-70 days), suggest that vaccination is not likely to be effective as a tool for control of H5N1 infection in meat duck flocks in the area.

Gram-negative bacteraemia; a multi-centre prospective evaluation of empiric antibiotic therapy and outcome in English acute hospitals.

Increasing antibiotic resistance makes choosing antibiotics for suspected Gram-negative infection challenging. This study set out to identify key determinants of mortality among patients with Gram-negative bacteraemia, focusing particularly on the importance of appropriate empiric antibiotic treatment. We conducted a prospective observational study of 679 unselected adults with Gram-negative bacteraemia at ten acute english hospitals between October 2013 and March 2014. Appropriate empiric antibiotic treatment was defined as intravenous treatment on the day of blood culture collection with an antibiotic to which the cultured organism was sensitive in vitro. Mortality analyses were adjusted for patient demographics, co-morbidities and illness severity. The majority of bacteraemias were community-onset (70%); most were caused by Escherichia coli (65%), Klebsiella spp. (15%) or Pseudomonas spp. (7%). Main foci of infection were urinary tract (51%), abdomen/biliary tract (20%) and lower respiratory tract (14%). The main antibiotics used were co-amoxiclav (32%) and piperacillin-tazobactam (30%) with 34% receiving combination therapy (predominantly aminoglycosides). Empiric treatment was inappropriate in 34%. All-cause mortality was 8% at 7 days and 15% at 30 days. Independent predictors of mortality (p <0.05) included older age, greater burden of co-morbid disease, severity of illness at presentation and inflammatory response. Inappropriate empiric antibiotic therapy was not associated with mortality at either time-point (adjusted OR 0.82; 95% CI 0.35-1.94 and adjusted OR 0.92; 95% CI 0.50-1.66, respectively). Although our study does not exclude an impact of empiric antibiotic choice on survival in Gram-negative bacteraemia, outcome is determined primarily by patient and disease factors.

Appropriate use of the Xpert® MTB/RIF assay in suspected tuberculous meningitis.

We disagree with the recommendation by the World Health Organization to use Xpert(®) MTB/RIF on cerebrospinal fluid for the initial diagnosis of tuberculous meningitis (TBM). TBM is a devastating disease requiring empirical treatment even when the probability of disease is low. We suggest that a useful TBM diagnostic test needs a negative predictive value (NPV) of ⩾ 99% so that empirical treatment can be stopped safely. The NPV of Xpert is around 84%, making a negative test of limited value. While better tests are awaited, a composite score, possibly combining Xpert with clinical variables and with high NPV, should be constructed and validated prospectively.

TLR9 gene region polymorphisms and susceptibility to tuberculosis in Vietnam.

Humans exposed to Mycobacterium tuberculosis (Mtb) show variation in susceptibility to infection and differences in tuberculosis (TB) disease outcome. Toll-like receptor 9 (TLR9) is a pattern recognition receptor that mediates recognition of Mtb and modulates Mtb-specific T-cell responses. Using a case-population design, we evaluated whether single nucleotide polymorphisms (SNPs) in the TLR9 gene region are associated with susceptibility to pulmonary or meningeal TB as well as neurologic presentation and mortality in the meningeal TB group. In a discovery cohort (n = 352 cases, 382 controls), three SNPs were associated with TB (all forms, p < 0.05) while three additional SNPs neared significance (0.05 < p < 0.1). When these six SNPs were evaluated in a validation cohort (n = 339 cases, 367 controls), one was significant (rs352142) while another neared significance (rs352143). When the cohorts were combined, rs352142 was most strongly associated with meningeal tuberculosis (dominant model; p = 0.0002, OR 2.36, CI 1.43-3.87) while rs352143 was associated with pulmonary tuberculosis (recessive model; p = 0.006, OR 5.3, CI 1.26-31.13). None of the SNPs were associated with mortality. This is the first demonstration of an association between a TLR9 gene region SNP and tuberculous meningitis. In addition, this extends previous findings that support associations of TLR9 SNPs with pulmonary tuberculosis.

A polymorphism in human CD1A is associated with susceptibility to tuberculosis.

CD1 proteins are antigen-presenting molecules that evolved to present lipids rather than peptides to T cells. However, unlike major histocompatibility complex genes, CD1 genes show low rates of polymorphism and have not been clearly associated with human disease. We report that an intronic polymorphism in CD1A (rs411089) is associated with susceptibility to tuberculosis in two cohorts of Vietnamese adults (combined cohort odds ratio 1.78; 95% confidence interval: 1.24-2.57; P=0.001). These data strengthen the hypothesis that CD1A-mediated lipid antigen presentation is important for controlling tuberculosis in humans.

Epiregulin (EREG) variation is associated with susceptibility to tuberculosis.

Although host genetics influences susceptibility to Mycobacterium tuberculosis, the human genes regulating pathogenesis remain largely unknown. We used M. tuberculosis-stimulated macrophage gene expression profiling in conjunction with a case-control genetic association study to discover epiregulin (EREG), as a novel candidate tuberculosis (TB) susceptibility gene. Using a genome-wide association study dataset, we found that among the 21 genes with greater than 50-fold induction, EREG had the most polymorphisms associated with TB. We genotyped haplotype-tagging polymorphisms in discovery (N = 337 cases, N = 380 controls) and validation (N = 332 cases) datasets and an EREG polymorphism (rs7675690) was associated with susceptibility to TB (genotypic comparison; corrected P = 0.00007). rs7675690 was also associated more strongly with infections caused by the Beijing lineage of M. tuberculosis when compared with non-Beijing strains (controls vs Beijing, OR 7.81, P = 8.7 × 10(-5); non-Beijing, OR 3.13, P = 0.074). Furthermore, EREG expression was induced in monocytes and peripheral blood mononuclear cells stimulated with M. tuberculosis as well as TLR4 and TLR2/1/6 ligands. In murine macrophages, EREG expression induced by M. tuberculosis was MYD88- and TLR2-dependent. Together, these data provide the first evidence for an important role for EREG as a susceptibility gene for human TB.

Effect of magnesium sulphate on urinary catecholamine excretion in severe tetanus.

Severe tetanus is characterised by muscle spasms and autonomic dysfunction. We recently reported the results of a randomised placebo controlled trial of magnesium sulphate infusions for the treatment of severe tetanus which showed magnesium was associated with improved muscle spasm and cardiovascular control. We hypothesised that magnesium controlled autonomic dysfunction by reducing catecholamine release and thus urinary excretion. Urinary adrenaline and noradrenaline concentrations were measured during the first 24 h of therapy in 180 adults with severe tetanus randomised to treatment with magnesium (n = 92) or placebo (n = 88). Magnesium therapy was associated with lower urinary adrenaline excretion and higher urinary noradrenaline excretion. High urinary adrenaline concentrations were associated with documented autonomic dysfunction. Patients given magnesium had significantly less autonomic dysfunction, required less cardiovascular stabilising drugs, and had lower urinary concentrations of adrenaline. These findings suggest adrenaline is important in the pathophysiology of severe tetanus and magnesium controls autonomic dysfunction by reducing adrenaline release.

A polymorphism in human TLR2 is associated with increased susceptibility to tuberculous meningitis.

Tuberculous meningitis (TBM) results from the haematogenous dissemination of Mycobacterium tuberculosis from the lung to the brain. Dissemination is believed to occur early during infection, before the development of adaptive immunity. Toll-like receptor 2 (TLR2) mediates recognition of M. tuberculosis and initiates the innate immune response to infection. We hypothesized that polymorphisms in the TLR2 gene influence bacterial dissemination and the development of TBM. A case-control study was designed to test the hypothesis. Cases of bacteriologically confirmed pulmonary tuberculosis (TB) (n=183) and TBM (n=175), and cord blood controls (n=389) were enrolled in Vietnam. TLR2 genotype 597CC was associated with susceptibility to TB (odds ratio (OR)=2.22, 95% confidence interval (CI): 1.23-3.99). The association was found with meningeal rather than pulmonary TB (TBM vs control, OR=3.26, 95% CI: 1.72-6.18), and was strongest when miliary TB was found on chest radiography (controls vs TBM with miliary TB, OR=5.28, 95% CI: 2.20-12.65). Furthermore, the association increased with the severity of neurologic symptoms (grade I TBM, OR=1.93, 95% CI: 0.54-6.92; grade II, OR=3.32, 95% CI: 0.84-13.2; and grade III, OR=5.70, 95% CI: 1.81-18.0). These results demonstrate a strong association of TLR2 SNP T597C with the development of TBM and miliary TB and indicate that TLR2 influences the dissemination of M. tuberculosis.

Molecular analysis of Mycobacterium tuberculosis causing multidrug-resistant tuberculosis meningitis.

SETTING: Tertiary referral hospitals in southern Vietnam. OBJECTIVE: Molecular characterisation of multidrug-resistant (MDR) tuberculous meningitis (TBM). DESIGN: Mycobacterium tuberculosis isolates from the cerebrospinal fluid (CSF) of 198 Vietnamese adults were compared with 237 isolates from patients with pulmonary tuberculosis (PTB) matched for age, sex and residential district. Isolates resistant to isoniazid or rifampicin (RMP) were sequenced in the rpoB and katG genes, inhA promoter and oxyR-ahpC intergenic regions. RESULTS: While drug resistance rates were lower in the CSF (2.5% MDR) than pulmonary isolates (5.9% MDR), the difference was not significant. The most commonly mutated codons were 531, 526 and 516 in rpoB and 315 in katG. Four novel triple mutants in rpoB were identified. CONCLUSION: RMP resistance is a good surrogate marker for MDR-TBM in this setting. However, probes directed against these three codons would have a maximum sensitivity of only 65%. A rapid phenotypic detection test may be more applicable for the diagnosis of MDR-TBM.

Magnesium sulphate for treatment of severe tetanus: a randomised controlled trial.

BACKGROUND: The most common cause of death in individuals with severe tetanus in the absence of mechanical ventilation is spasm-related respiratory failure, whereas in ventilated patients it is tetanus-associated autonomic dysfunction. Our aim was to determine whether continuous magnesium sulphate infusion reduces the need for mechanical ventilation and improves control of muscle spasms and autonomic instability. METHODS: We did a randomised, double blind, placebo controlled trial in 256 Vietnamese patients over age 15 years with severe tetanus admitted to the Hospital for Tropical Medicine, Ho Chi Minh City, Vietnam. Participants were randomly assigned magnesium sulphate (n=97) or placebo solution (n=98) intravenously for 7 days. The primary outcomes were requirement of assisted ventilation and of drugs to control muscle spasms and cardiovascular instability within the 7-day study period. Analyses were done by intention to treat. This trial is registered as an International Standard Randomised Clinical Trial, number ISRCTN74651862. FINDINGS: No patients were lost to follow-up. There was no difference in requirement for mechanical ventilation between individuals treated with magnesium and those receiving placebo (odds ratio 0.71, 95% CI 0.36-1.40; p=0.324); survival was also much the same in the two groups. However, compared with the placebo group, patients receiving magnesium required significantly less midazolam (7.1 mg/kg per day [0.1-47.9] vs 1.4 mg/kg per day [0.0-17.3]; p=0.026) and pipecuronium (2.3 mg/kg per day [0.0-33.0] vs 0.0 mg/kg per day [0.0-14.8]; p=0.005) to control muscle spasms and associated tachycardia. Individuals receiving magnesium were 4.7 (1.4-15.9) times less likely to require verapamil to treat cardiovascular instability than those in the placebo group. The incidence of adverse events was not different between the groups. INTERPRETATION: Magnesium infusion does not reduce the need for mechanical ventilation in adults with severe tetanus but does reduce the requirement for other drugs to control muscle spasms and cardiovascular instability.

Pyrexia of unknown origin.

Classical pyrexia of unknown origin (PUO), defined as fever of >38 °C on several occasions for greater than three weeks despite investigation in hospital (>3 days) or out of hospital (>2 visits), is an uncommon but challenging problem. The incidence and aetiology vary according to the geographic region, the age structure of the population, and the immune status of the patient; alternative definitions of PUO exist for immune compromised individuals. Preliminary investigations should be determined by detailed history and repeated examination. Biopsy of abnormal tissues should be performed early. If uncertainty persists, abdominal computerised tomography (CT), radiolabeled white cell scans, and the Duke endocarditis criteria carry the highest diagnostic yield. Blind bone marrow biopsy is probably only useful in immunocompromised patients.