Functional genetic variation in pe/ppe genes contributes to diversity in Mycobacterium tuberculosis lineages and potential interactions with the human host.

INTRODUCTION: Around 10% of the coding potential of Mycobacterium tuberculosisis constituted by two poorly understood gene families, the pe and ppe loci, thought to be involved in host-pathogen interactions. Their repetitive nature and high GC content have hindered sequence analysis, leading to exclusion from whole-genome studies. Understanding the genetic diversity of pe/ppe families is essential to facilitate their potential translation into tools for tuberculosis prevention and treatment. METHODS: To investigate the genetic diversity of the 169 pe/ppe genes, we performed a sequence analysis across 73 long-read assemblies representing seven different lineages of M. tuberculosis and M. bovis BCG. Individual pe/ppe gene alignments were extracted and diversity and conservation across the different lineages studied. RESULTS: The pe/ppe genes were classified into three groups based on the level of protein sequence conservation relative to H37Rv, finding that >50% were conserved, with indels in pe_pgrs and ppe_mptr sub-families being major drivers of structural variation. Gene rearrangements, such as duplications and gene fusions, were observed between pe and pe_pgrs genes. Inter-lineage diversity revealed lineage-specific SNPs and indels. DISCUSSION: The high level of pe/ppe genes conservation, together with the lineage-specific findings, suggest their phylogenetic informativeness. However, structural variants and gene rearrangements differing from the reference were also identified, with potential implications for pathogenicity. Overall, improving our knowledge of these complex gene families may have insights into pathogenicity and inform the development of much-needed tools for tuberculosis control

Etiology of Pediatric Bacterial Meningitis Pre- and Post-PCV13 Introduction Among Children Under 5 Years Old in Lomé, Togo.

BACKGROUND: Pediatric bacterial meningitis (PBM) causes severe morbidity and mortality within Togo. Thus, as a member of the World Health Organization coordinated Invasive Bacterial Vaccine Preventable Diseases network, Togo conducts surveillance targeting Streptococcus pneumoniae (pneumococcus), Neisseria meningitidis (meningococcus), and Haemophilus influenzae, at a sentinel hospital within the capital city, Lomé, in the southernmost Maritime region. METHODS: Cerebrospinal fluid was collected from children <5 years with suspected PBM admitted to the Sylvanus Olympio Teaching Hospital. Phenotypic detection of pneumococcus, meningococcus, and H. influenzae was confirmed through microbiological techniques. Samples were shipped to the Regional Reference Laboratory to corroborate results by species-specific polymerase chain reaction. RESULTS: Overall, 3644 suspected PBM cases were reported, and 98 cases (2.7%: 98/3644) were confirmed bacterial meningitis. Pneumococcus was responsible for most infections (67.3%: 66/98), followed by H. influenzae (23.5%: 23/98) and meningococcus (9.2%: 9/98). The number of pneumococcal meningitis cases decreased by 88.1% (52/59) postvaccine introduction with 59 cases from July 2010 to June 2014 and 7 cases from July 2014 to June 2016. However, 5 cases caused by nonvaccine serotypes were observed. Fewer PBM cases caused by vaccine serotypes were observed in infants <1 year compared to children 2-5 years. CONCLUSIONS: Routine surveillance showed that PCV13 vaccination is effective in preventing pneumococcal meningitis among children <5 years of age in the Maritime region. This complements the MenAfriVac vaccination against meningococcal serogroup A to prevent meningitis outbreaks in the northern region of Togo. Continued surveillance is vital for estimating the prevalence of PBM, determining vaccine impact, and anticipating epidemics in Togo

Host-Directed Therapies for tackling Multi-Drug Resistant TB – learning from the Pasteur-Bechamp debates

Tuberculosis (TB) remains a global emergency causing an estimated 1.5 million deaths annually. For several decades the major focus of TB treatment has been on antibiotic development targeting Mycobacterium tuberculosis (M.tb). The lengthy TB treatment duration and poor treatment outcomes associated with multi-drug resistant TB (MDR-TB) are of major concern. The sparse new TB drug pipeline and widespread emergence of MDR-TB signal an urgent need for more innovative interventions to improve treatment outcomes. Building on the historical Pasteur-Bechamp debates on the role of the ‘microbe’ versus the ‘host internal milieu’ in disease causation, we make the case for parallel investments into host-directed therapies (HDTs). A range of potential HDTs are now available which require evaluation in randomized controlled clinical trials as adjunct therapies for shortening the duration of TB therapy and improving treatment outcomes for drug-susceptible TB and MDR-TB. Funder initiatives that may enable further research into HDTs are described

Towards host-directed therapies for tuberculosis

The treatment of tuberculosis is based on combinations of drugs that directly target Mycobacterium tuberculosis. A new global initiative is now focusing on a complementary approach of developing adjunct host-directed therapies. Despite the availability of effective antibiotics for tuberculosis (TB) for the past half century, it remains an important global health problem; there are ~9 million active TB cases and ~1.5 million TB-induced deaths per year (see the World Health Organization (WHO) Global Tuberculosis Report in Further information). Health services around the world face major barriers to achieving optimal outcomes from current TB treatment regimens. These barriers include: the spread of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB); complex and toxic treatment regimens for MDR-TB; HIV co-infection; pharmacokinetic interactions between TB drugs and antiretroviral drugs; relapse; permanent damage to lung and other tissues; long-term functional disability; immune reconstitution inflammatory syndrome (IRIS); and co-morbidity with non-communicable diseases such as diabetes and chronic obstructive airway diseases. Another fundamental problem is the long duration of TB drug treatment (6 months for drug-sensitive TB and at least 18 months for drug-resistant TB) to achieve a cure, owing to the presence of dormant Mycobacterium tuberculosis bacilli that are phenotypically resistant to current classes of anti-TB drugs, which can only target bacterial replication. There is therefore an urgent need for new TB treatments. However, the TB drug pipeline is thin1, 2. For the past 60 years, efforts to develop new treatments have focused on compounds and regimens that target M. tuberculosis directly. Recently, however, attention has focused on investigating a range of adjunct treatment interventions known as host-directed therapies (HDTs) that instead target the host response to infection. Here, we highlight the rationale for HDTs, the current portfolio of HDTs and their mechanisms of action, and a consortium-based approach to drive forward their evaluation in clinical trials

Host and pathogen factors that influence variability of Mycobacterium tuberculosis lipid body content in sputum from patients with tuberculosis: an observational study

Background High proportions of Mycobacterium tuberculosis cells in sputum containing triacylglycerol-rich lipid bodies have been shown to be associated with treatment failure or relapse following antituberculous chemotherapy. Although lipid body determination is a potential biomarker for supporting clinical trial and treatment decisions, factors influencing variability in sputum frequencies of lipid body-positive (%LB+) M tuberculosis in patients are unknown. We aimed to test our hypothesis that exposure to host-generated NO and M tuberculosis strains are factors associated with differences in sputum %LB+. Methods In this observational study, we determined %LB+ frequencies before treatment by microscopy in patients with smear-positive tuberculosis from two separate prospective observational study settings (Gondar, Ethiopia, recruited between May 1, 2010, and April 30, 2011, and Fajara, The Gambia, who provided sputum samples before treatment between May 5, 2010, and Dec 22, 2011). In Ethiopia, fractional exhaled nitric oxide (FeNO) was measured as a biomarker of host NO, and M tuberculosis strain differences were determined by spoligotyping. Treatment response was assessed by percentage weight change after 7 months. In The Gambia, treatment responses were assessed as change in BMI and radiographic burden of disease after 6 months. Sputum M tuberculosis isolates were studied in vitro for their %LB+ and triacylglycerol synthase 1 (tgs1) mRNA responses to NO exposure. Propidium iodide staining was used as a measure of NO strain toxicity. Correlation between in vitro %LB+ frequencies following NO exposure and those of the same strain in sputum was examined with linear regression and Dunnett’s multiple comparison test. Findings In Ethiopia, 73 patients who were smear positive for pulmonary tuberculosis were recruited (43 [59%] were male and 30 [41%] were female). Of these, the %LB+ in the sputum of 59 patients showed linear correlation with log10 FeNO (r2=0·28; p Interpretation M tuberculosis strain and exposure to host-generated NO are associated with sputum %LB+. Our results support the use of M tuberculosis strain-dependent sputum %LB+ as a predictive biomarker of treatment response.</p

Kinetics of gene expression in <i>Maf-</i> and <i>Mtb</i>-infected groups following treatment.

<p>The expression kinetics of genes that showed significant differences between <i>Maf-</i> and <i>Mtb</i>-infected groups following overnight incubation with Medium alone (A), ESAT-6/CFP-10 (B) and live <i>Maf</i> (C). Line plots show the predicted mean and its 95% CI of log2 transformed gene expression levels in <i>Maf-</i> and <i>Mtb</i>-infected groups at 0, 2 and 6 months of treatment. Wald test through contrasts analysis following a random-intercept model adjusted for age, sex and ethnicity was used to assess interaction between lineage group and time point on gene expression. The legend shows <i>Maf-infected</i> group (dashed lines) and <i>Mtb-infected</i> group (solid lines) respectively. P-values of the interactions are shown.</p

Demographic, clinical and haematology characteristics of <i>M</i>. <i>africanum</i> and <i>M</i>. <i>tuberculosis</i> patients.

<p>Demographic, clinical and haematology characteristics of <i>M</i>. <i>africanum</i> and <i>M</i>. <i>tuberculosis</i> patients.</p

Differential cytokine production between <i>Maf-</i> and <i>Mtb</i>-infected patients before and after treatment.

<p>Whole blood incubated overnight with medium only (I), revealed differences in the concentrations of IL12p70 (A), PDGF-ββ (B), MIP-1α (C), IL-15 (D) and IL-8 (E) between <i>Mtb-</i> and <i>Maf</i>-infected patients at 6 month of treatment. (II) Only ESAT-6/CFP-10 stimulation induced significant differences in cytokine production between <i>Mtb-</i> and <i>Maf</i>-infected patients above the background level of IFN-γ (A), IL-2 (B), IL-1ra (C), TNF-α (D), GM-CSF (E) and Rantes (F). Dot plots show log-2 transformed cytokine concentrations measured with Bio-Plex assay. Horizontal bars indicate median cytokine concentration by lineage groups, <i>Maf</i>-infected patients (closed circles, n = 26 and 20) and <i>Mtb</i>-infected patients (open circles, n = 49 and 31) respectively at 0 and 6 months of TB treatment. Log-2 transformed cytokine concentrations were compared between lineage groups using a random-intercept model adjusted for age, sex and ethnicity, and Sidak multiple comparison correction. Contrasts analysis was used after estimation to compute the difference in each cytokine concentration between lineage groups and used Wald test for assessing the significance at each time point. P-values of the differences are shown.</p

Gene expression profiles differ between <i>Maf-</i> and <i>Mtb</i>-infected patients before and after treatment.

<p>dcRT-MLPA was performed on RNA extracted from whole blood incubated overnight with medium only (A), ESAT-6/CFP-10 (B) and live <i>Maf</i> (C). Median gene expression levels (peak areas normalized to GAPDH and log2 transformed) of the indicated genes are shown in box-and-whisker plots. Equal number of samples were analysed at 0 and 6 months of treatment in each group of <i>Maf</i>-infected (n = 20; white boxes) and <i>Mtb</i>-infected (n = 31; grey boxes) patients, respectively. Log-2 transformed gene expression data were compared between the groups using contrast analysis following a random-intercept model adjusted for age, sex and ethnicity, and Sidak multiple comparison correction. Wald test was used to assess the significance at each time point. P-values of significant differences are shown.</p