Pre-treatment cerebrospinal fluid bacterial load correlates with inflammatory response and predicts neurological events during tuberculous meningitis treatment.

Background Mycobacterium tuberculosis (Mtb) bacillary load in the brain of those with tuberculous meningitis (TBM) may reflect the host ability to control the pathogen and determine disease severity and treatment outcomes. Methods We measured pre-treatment cerebrospinal fluid (CSF) Mtb bacterial load by GeneXpert in 692 adults with TBM. We sought to understand the relationship between CSF bacterial load and inflammation, and their respective impact on disease severity and treatment outcomes. Results Ten-fold higher Mtb load was associated with increased disease severity (Odds Ratio=1.59, p=0.001 for grade 1 versus grade 3), and increased CSF neutrophils (r=0.364, p<0.0001) and cytokine concentrations (r=0.438, p<0.0001). High Mtb load predicted new neurological events after starting treatment (Multinomial logistic regression, p=0.005), but not death. Death was previously associated with attenuated inflammatory response at the start of treatment, with reduced cytokine concentrations compared to survivors. In contrast, patients with high pre-treatment CSF bacterial loads, cytokines, and neutrophils were more likely to subsequently suffer neurological events. Conclusions Pre-treatment GeneXpert-derived Mtb load may be a useful predictor of neurological complications occurring during TBM treatment. Therapeutic strategies aimed at reducing neurological complications and deaths from TBM may need reassessment, given the evidence for their divergent pathogenesis

A robust SNP barcode for typing Mycobacterium tuberculosis complex strains

Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type

Randomized pharmacokinetic and pharmacodynamic comparison of fluoroquinolones for tuberculous meningitis.

Tuberculous meningitis (TBM) is the most lethal form of tuberculosis, and new treatments that improve outcomes are required. We randomly assigned adults with TBM to treatment with standard antituberculosis treatment alone or in combination with ciprofloxacin (750 mg/12 h), levofloxacin (500 mg/12 h), or gatifloxacin (400 mg/24 h) for the first 60 days of therapy. Fluoroquinolone concentrations were measured with plasma and cerebrospinal fluid (CSF) specimens taken at predetermined, randomly assigned times throughout treatment. We aimed to describe the pharmacokinetics of each fluoroquinolone during TBM treatment and evaluate the relationship between drug exposure and clinical response over 270 days of therapy (Controlled Trials number ISRCTN07062956). Sixty-one patients with TBM were randomly assigned to treatment with no fluoroquinolone (n = 15), ciprofloxacin (n = 16), levofloxacin (n = 15), or gatifloxacin (n = 15). Cerebrospinal fluid penetration, measured by the ratio of the plasma area under the concentration-time curve from 0 to 24 h (AUC0–24) to the cerebrospinal fluid AUC0–24, was greater for levofloxacin (median, 0.74; range, 0.58 to 1.03) than for gatifloxacin (median, 0.48; range, 0.47 to 0.50) or ciprofloxacin (median, 0.26; range, 0.11 to 0.77). Univariable and multivariable analyses of fluoroquinolone exposure against a range of different treatment responses revealed worse outcomes among patients with lower and higher plasma and CSF exposures than for patients with intermediate exposures (a U-shaped exposure-response). TBM patients most likely to benefit from fluoroquinolone therapy were identified, along with exposure-response relationships associated with improved outcomes. Fluoroquinolones add antituberculosis activity to the standard treatment regimen, but to improve outcomes of TBM, they must be started early, before the onset of coma

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

BACKGROUND: Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. METHODS AND FINDINGS: We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42-4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43-5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. CONCLUSIONS: We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian reg

Sources of Multidrug Resistance in Patients With Previous Isoniazid-Resistant Tuberculosis Identified Using Whole Genome Sequencing: A Longitudinal Cohort Study

Background Meta-analysis of patients with isoniazid-resistant tuberculosis given standard first-line anti-tuberculosis treatment indicated an increased risk of multi-drug resistant tuberculosis (MDR-TB) emerging (8%), compared to drug-sensitive tuberculosis (0.3%). Here we use whole genome sequencing (WGS) to investigate whether treatment of patients with pre-existing isoniazid resistant disease with first-line anti-tuberculosis therapy risks selecting for rifampicin resistance, and hence MDR-TB. Methods Patients with isoniazid-resistant pulmonary TB were recruited and followed up for 24 months. Drug-susceptibility testing was performed by Microscopic observation drug-susceptibility assay (MODS), Mycobacterial Growth Indicator Tube (MGIT) and by WGS on isolates at first presentation and in the case of re-presentation. Where MDR-TB was diagnosed, WGS was used to determine the genomic relatedness between initial and subsequent isolates. De novo emergence of MDR-TB was assumed where the genomic distance was five or fewer single nucleotide polymorphisms (SNPs) whereas reinfection with a different MDR-TB strain was assumed where the distance was 10 or more SNPs. Results 239 patients with isoniazid-resistant pulmonary tuberculosis were recruited. Fourteen (14/239, 5.9%) patients were diagnosed with a second episode of tuberculosis that was multi-drug resistant. Six (6/239, 2.5%) were identified as having evolved MDR-TB de novo and six as having been re-infected with a different strain. In two cases the genomic distance was between 5-10 SNPs and therefore indeterminate. Conclusions In isoniazid-resistant TB, de novo emergence and reinfection of MDR-TB strains equally contributed to MDR development. Early diagnosis and optimal treatment of isoniazid resistant TB are urgently needed to avert the de novo emergence of MDR-TB during treatment

Standardized methods for enhanced quality and comparability of tuberculous meningitis studies

Tuberculous meningitis remains a major cause of death and disability in tuberculosis endemic areas, especially in young children and immunocompromised adults. Research aimed at improving outcomes is hampered by poor standardization, which limits study comparison and the generalizability of results. We propose standardized methods for the conduct of tuberculous meningitis clinical research that were drafted at an international tuberculous meningitis research meeting organized by the Oxford University Clinical Research Unit in Vietnam. We propose a core dataset including demographic and clinical information to be collected at study enrolment, important aspects related to patient management and monitoring, and standardized reporting of patient outcomes. The criteria proposed for the conduct of observational and intervention tuberculous meningitis studies should improve the quality of future research outputs, facilitate multi-centre studies and meta-analyses of pooled data, and could provide the foundation for a global tuberculous meningitis data repository

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 β-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 PT

E-learning repository system for sharing learning resources among Saudi universities

This paper discusses the status and diversity of needs for building a centralized e-learning repository system for Saudi Universities. The study is based on surveys that were distributed to faculty members in various Saudi Universities. The purpose is to provide an analytical overview of the current needs for a unified e-learning repository system among Saudi Universities for sharing learning objects and materials. Moreover, the primary aim of the study is to give an evaluation of the needs of faculty members by gathering facts about the current demands and future adoption among Saudi Universities. To achieve this, the services needed by each part in the universities were analyzed