The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis.

The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193-0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15-2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis.

Relationship between Mycobacterium tuberculosis genotype and the clinical phenotype of pulmonary and meningeal tuberculosis.

We used large sequence polymorphisms to determine the genotypes of 397 isolates of Mycobacterium tuberculosis from human immunodeficiency virus-uninfected Vietnamese adults with pulmonary (n = 235) or meningeal (n = 162) tuberculosis. We compared the pretreatment radiographic appearances of pulmonary tuberculosis and the presentation, response to treatment, and outcome of tuberculous meningitis between the genotypes. Multivariate analysis identified variables independently associated with genotype and outcome. A higher proportion of adults with pulmonary tuberculosis caused by the Euro-American genotype had consolidation on chest X-ray than was the case with disease caused by other genotypes (P = 0.006). Multivariate analysis revealed that meningitis caused by the East Asian/Beijing genotype was independently associated with a shorter duration of illness before presentation and fewer cerebrospinal fluid (CSF) leukocytes. Older age, fewer CSF leukocytes, and the presence of hemiplegia (but not strain lineage) were independently associated with death or severe disability, although the East Asian/Beijing genotype was strongly associated with drug-resistant tuberculosis. The genotype of M. tuberculosis influenced the presenting features of pulmonary and meningeal tuberculosis. The association between the East Asian/Beijing lineage and disease progression and CSF leukocyte count suggests the lineage may alter the presentation of meningitis by influencing the intracerebral inflammatory response. In addition, increased drug resistance among bacteria of the East Asian/Beijing lineage might influence the response to treatment. This study suggests the genetic diversity of M. tuberculosis has important clinical consequences.

Genomic survey of Clostridium difficile reservoirs in the East of England implicates environmental contamination of wastewater treatment plants by clinical lineages.

There is growing evidence that patients with Clostridiumdifficile-associated diarrhoea often acquire their infecting strain before hospital admission. Wastewater is known to be a potential source of surface water that is contaminated with C. difficile spores. Here, we describe a study that used genome sequencing to compare C. difficile isolated from multiple wastewater treatment plants across the East of England and from patients with clinical disease at a major hospital in the same region. We confirmed that C. difficile from 65 patients were highly diverse and that most cases were not linked to other active cases in the hospital. In total, 186 C. difficile isolates were isolated from effluent water obtained from 18 municipal treatment plants at the point of release into the environment. Whole genome comparisons of clinical and environmental isolates demonstrated highly related populations, and confirmed extensive release of toxigenic C. difficile into surface waters. An analysis based on multilocus sequence types (STs) identified 19 distinct STs in the clinical collection and 38 STs in the wastewater collection, with 13 of 44 STs common to both clinical and wastewater collections. Furthermore, we identified five pairs of highly similar isolates (≤2 SNPs different in the core genome) in clinical and wastewater collections. Strategies to control community acquisition should consider the need for bacterial control of treated wastewater.

HIV-1 drug resistance in antiretroviral-naive individuals with HIV-1-associated tuberculous meningitis initiating antiretroviral therapy in Vietnam.

BACKGROUND: Access to antiretroviral therapy (ART) for HIV-infected individuals in Vietnam is rapidly expanding, but there are limited data on HIV drug resistance (HIVDR) to guide ART strategies. METHODS: We retrospectively conducted HIVDR testing in 220 ART-naive individuals recruited to a randomized controlled trial of immediate versus deferred ART in individuals with HIV-associated tuberculous meningitis in Ho Chi Minh City (HCMC) from 2005-2008. HIVDR mutations were identified by population sequencing of the HIV pol gene and were defined based on 2009 WHO surveillance drug resistance mutations (SDRMs). RESULTS: We successfully sequenced 219/220 plasma samples of subjects prior to ART; 218 were subtype CRF01_AE and 1 was subtype B. SDRMs were identified in 14/219 (6.4%) subjects; 8/14 were resistant to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs; T69D, L74V, V75M, M184V/I and K219R), 5/14 to non-nucleoside reverse transcriptase inhibitors (NNRTIs; K103N, V106M, Y181C, Y188C and G190A), 1/14 to both NRTIs and NNRTIs (D67N and Y181C) and none to protease inhibitors. After 6 months of ART, eight subjects developed protocol-defined virological failure. HIVDR mutations were identified in 5/8 subjects. All five had mutations with high-level resistance to NNRTIs and three had mutations with high-level resistance to NRTIs. Due to a high early mortality rate (58%), the effect of pre-existing HIVDR mutations on treatment outcome could not be accurately assessed. CONCLUSIONS: The prevalence of WHO SDRMs in ART-naive individuals with HIV-associated tuberculous meningitis in HCMC from 2005-2008 is 6.4%. The SDRMs identified conferred resistance to NRTIs and/or NNRTIs, reflecting the standard first-line ART regimens in Vietnam.

Evaluation of the MODS culture technique for the diagnosis of tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is a devastating condition. The rapid instigation of appropraite chemotherapy is vital to reduce morbidity and mortality. However rapid diagnosis remains elusive; smear microscopy has extremely low sensitivity on cerebrospinal fluid (CSF) in most laboratories and PCR requires expertise with advanced infrastructure and has sensitivity of only around 60% under optimal conditions. Neither technique allows for the microbiological isolation of M. tuberculosis and subsequent drug susceptibility testing. We evaluated the recently developed microscopic observation drug susceptibility (MODS) assay format for speed and accuracy in diagnosing TBM. METHODOLOGY/PRINCIPAL FINDINGS: Two hundred and thirty consecutive CSF samples collected from 156 patients clinically suspected of TBM on presentation at a tertiary referal hospital in Vietnam were enrolled into the study over a five month period and tested by Ziehl-Neelsen (ZN) smear, MODS, Mycobacterial growth Indicator tube (MGIT) and Lowenstein-Jensen (LJ) culture. Sixty-one samples were from patients already on TB therapy for >1day and 19 samples were excluded due to untraceable patient records. One hundred and fifty samples from 137 newly presenting patients remained. Forty-two percent (n = 57/137) of patients were deemed to have TBM by clinical diagnostic and microbiological criteria (excluding MODS). Sensitivity by patient against clinical gold standard for ZN smear, MODS MGIT and LJ were 52.6%, 64.9%, 70.2% and 70.2%, respectively. Specificity of all microbiological techniques was 100%. Positive and negative predictive values for MODS were 100% and 78.7%, respectively for HIV infected patients and 100% and 82.1% for HIV negative patients. The median time to positive was 6 days (interquartile range 5-7), significantly faster than MGIT at 15.5 days (interquartile range 12-24), and LJ at 24 days (interquartile range 18-35 days) (P<0.01). CONCLUSIONS: We have shown MODS to be a sensitive, rapid technique for the diagnosis of TBM with high sensitivity, ease of performance and low cost (0.53 USD/sample).

Pretreatment intracerebral and peripheral blood immune responses in Vietnamese adults with tuberculous meningitis: diagnostic value and relationship to disease severity and outcome.

Tuberculous meningitis (TBM) is the most devastating form of tuberculosis. Both intracerebral and peripheral blood immune responses may be relevant to pathogenesis, diagnosis, and outcome. In this study, the relationship between pretreatment host response, disease phenotype, and outcome in Vietnamese adults with TBM was examined. Before treatment, peripheral blood IFN-gamma ELISPOT responses to the Mycobacterium tuberculosis Ags ESAT-6, CFP-10, and purified protein derivative (PPD) were a poor diagnostic predictor of TBM. Cerebrospinal fluid IL-6 concentrations at presentation were independently associated with severe disease presentation, suggesting an immunological correlate of neurological damage before treatment. Surprisingly however, elevated cerebrospinal fluid inflammatory cytokines were not associated with death or disability in HIV-negative TBM patients at presentation. HIV coinfection attenuated multiple cerebrospinal fluid inflammatory indices. Low cerebrospinal fluid IFN-gamma concentrations were independently associated with death in HIV-positive TBM patients, implying that IFN-gamma contributes to immunity and survival. Collectively, these results reveal the effect of HIV coinfection on the pathogenesis of TBM and suggest that intracerebral immune responses, at least in HIV-negative cases, may not be as intimately associated with disease outcome as previously thought.