Reinout van Crevel

Possible underlying mechanisms for successful emergence of the Mycobacterium tuberculosis Beijing genotype strains

The wide geographic distribution of one clade of Mycobacterium tuberculosis, the Beijing genotype family, and its genetic homogeneity, suggests that strains belonging to this grouping might have a selective advantage over other M tuberculosis strains. This hypothesis was addressed by reviewing molecular-epidemiological, experimental, and clinical studies. Beijing strains represent about 50% of strains in east Asia and at least 13% of strains worldwide. Their emergence might be linked to escape from BCG vaccination, and to multidrug resistance, which is associated with the Beijing genotype in many areas. Different animal models have shown Beijing strains to be more virulent, and to cause more histopathological changes, higher outgrowth, and increased mortality. At a molecular level, Beijing strains have specific properties in terms of protein and lipid structures and their interaction with the human immune system. Finally, the Beijing genotype has been linked to polymorphisms in immune genes, suggesting the possibility of human-mycobacterial co-evolution. The emergence of the Beijing genotype family might represent an evolutionary response of M tuberculosis to vaccination or antibiotic treatment, with an important negative impact on tuberculosis control. More research is needed to further unravel the mechanisms underlying the emergence of M tuberculosis Beijing genotype strains, and examine the implications for future control strategies.

Intensified regimen containing rifampicin and moxifloxacin for tuberculous meningitis: an open-label, randomised controlled phase 2 trial.

BACKGROUNDnIntensified antibiotic treatment might improve the outcome of tuberculous meningitis. We assessed pharmacokinetics, safety, and survival benefit of several treatment regimens containing high-dose rifampicin and moxifloxacin in patients with tuberculous meningitis in a hospital setting.nnnMETHODSnIn an open-label, phase 2 trial with a factorial design in one hospital in Indonesia, patients (aged >14 years) with tuberculous meningitis were randomly assigned to receive, according to a computer-generated schedule, first rifampicin standard dose (450 mg, about 10 mg/kg) orally or high dose (600 mg, about 13 mg/kg) intravenously, and second oral moxifloxacin 400 mg, moxifloxacin 800 mg, or ethambutol 750 mg once daily. All patients were given standard-dose isoniazid, pyrazinamide, and adjunctive corticosteroids. After 14 days of treatment all patients continued with standard treatment for tuberculosis. Endpoints included pharmacokinetic analyses of the blood and cerebrospinal fluid, adverse events attributable to tuberculosis treatment, and survival. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01158755.nnnFINDINGSn60 patients were randomly assigned to receive rifampicin standard dose (12 no moxifloxacin, ten moxifloxacin 400 mg, and nine moxifloxacin 800 mg) and high dose (ten no moxifloxacin, nine moxifloxacin 400 mg, and ten moxifloxacin 800 mg). A 33% higher dose of rifampicin, intravenously, led to a three times higher geometric mean area under the time-concentration curve up to 6 h after dose (AUC(0-6); 78·7 mg.h/L [95% CI 71·0-87·3] vs 26·0 mg.h/L [19·0-35·6]), maximum plasma concentrations (C(max); 22·1 mg/L [19·9-24·6] vs 6·3 mg/L [4·9-8·3]), and concentrations in cerebrospinal fluid (0·60 mg/L [0·46-0·78] vs 0·21 mg/L [0·16-0·27]). Doubling the dose of moxifloxacin resulted in a proportional increase in plasma AUC(0-6) (31·5 mg.h/L [24·1-41·1] vs 15·1 mg.h/L [12·8-17·7]), C(max) (7·4 mg/L [5·6-9·6] vs 3·9 mg/L [3·2-4·8]), and drug concentrations in the cerebrospinal fluid (2·43 mg/L [1·81-3·27] vs 1·52 mg/L [1·28-1·82]). Intensified treatment did not result in increased toxicity. 6 month mortality was substantially lower in patients given high-dose rifampicin intravenously (ten [35%] vs 20 [65%]), which could not be explained by HIV status or severity of disease at the time of presentation (adjusted HR 0·42; 95% CI 0·20-0·91; p=0·03).nnnINTERPRETATIONnThese data suggest that treatment containing a higher dose of rifampicin and standard-dose or high-dose moxifloxacin during the first 2 weeks is safe in patients with tuberculous meningitis, and that high-dose intravenous rifampicin could be associated with a survival benefit in patients with severe disease.nnnFUNDINGnRoyal Dutch Academy of Arts and Sciences, Netherlands Foundation for Scientific Research, and Padjadjaran University, Bandung, Indonesia.

Exposure to Rifampicin Is Strongly Reduced in Patients with Tuberculosis and Type 2 Diabetes

BACKGROUNDnType 2 diabetes (DM) is a strong risk factor for tuberculosis (TB) and is associated with a slower response to TB treatment and a higher mortality rate. Because lower concentrations of anti-TB drugs may be a contributing factor, we compared the pharmacokinetics of rifampicin in patients with TB, with and without DM.nnnMETHODSnSeventeen adult Indonesian patients with TB and DM and 17 age- and sex-matched patients with TB and without DM were included in the study during the continuation phase of TB treatment. All patients received 450 mg of rifampicin (10 mg/kg) and 600 mg of isoniazid 3 times weekly. Steady-state plasma concentrations of rifampicin and its metabolite desacetylrifampicin were assessed at 0, 2, 4, and 6 h after drug intake.nnnRESULTSnGeometric means of rifampicin exposure (AUC(0-6 h)) were 12.3 mg x h/L (95% confidence interval [CI], 8.0-24.2) in patients with TB and DM, and 25.9 mg x h/L (95% CI, 21.4-40.2) in patients with TB only (P=.003). Similar differences were found for the maximum concentration of rifampicin. No significant differences in time to maximum concentration of rifampicin were observed. The AUC(0-6 h) of desacetylrifampicin was also much lower in patients with TB and DM versus patients with TB only (geometric mean, 0.60 vs. 3.2 mg x h/L; P=.001). Linear regression analysis revealed that higher body weight (P<.001), the presence of DM (P=.06), and plasma glucose concentration (P=.016) were correlated with exposure to rifampicin.nnnCONCLUSIONnExposure (AUC(0-6 h)) to rifampicin was 53% lower in Indonesian patients with TB and DM, compared with patients with TB only. Patients with TB and DM who have a higher body weight may need a higher dose of rifampicin.

Implications of the global increase of diabetes for tuberculosis control and patient care

Objectivesu2002 To review the current knowledge about tuberculosis (TB) and diabetes, assessing the implication of the global increase of diabetes for TB control and patient care.

Mycobacterium tuberculosis induces IL-17A responses through TLR4 and dectin-1 and is critically dependent on endogenous IL-1

In the present study, we dissected the pathways that trigger the IL‐17A responses by MTB. Dectin‐1 and TLR4 were shown to be involved in MTB‐induced IL‐17A production, and blockade of the NOD2, TLR2, or MR had no effect on IL‐17A. The MAPK Erk, known to mediate transcription of IL‐1β mRNA, was strongly involved in the IL‐17A production induced by MTB. The intracellular enzymes caspase‐1 and serine proteases, which process pro‐IL‐1β into the active IL‐1β, were also crucial for the induction of IL‐17A. Lastly, the MTB‐induced IL‐17A response was strongly dependent on signaling through the IL‐1R but not the IL‐6R pathway. In conclusion, the MTB‐induced IL‐17A response relies strongly on the endogenous IL‐1 pathway and IL‐1R signaling. TLR4 and dectin‐1 are the main receptors responsible for mediating the signals responsible for IL‐17A production by MTB. These findings contribute to a better understanding of the host response to mycobacteria and provide the opportunity to explore potential, novel, therapeutic strategies against TB.

A genome wide association study of pulmonary tuberculosis susceptibility in Indonesians

BackgroundThere is reason to expect strong genetic influences on the risk of developing active pulmonary tuberculosis (TB) among latently infected individuals. Many of the genome wide linkage and association studies (GWAS) to date have been conducted on African populations. In order to identify additional targets in genetically dissimilar populations, and to enhance our understanding of this disease, we performed a multi-stage GWAS in a Southeast Asian cohort from Indonesia.MethodsIn stage 1, we used the Affymetrix 100 K SNP GeneChip marker set to genotype 259 Indonesian samples. After quality control filtering, 108 cases and 115 controls were analyzed for association of 95,207 SNPs. In stage 2, we attempted validation of 2,453 SNPs with promising associations from the first stage, in 1,189 individuals from the same Indonesian cohort, and finally in stage 3 we selected 251 SNPs from this stage to test TB association in an independent Caucasian cohort (n = 3,760) from Russia.ResultsOur study suggests evidence of association (P = 0.0004-0.0067) for 8 independent loci (nominal significance P < 0.05), which are located within or near the following genes involved in immune signaling: JAG1, DYNLRB2, EBF1, TMEFF2, CCL17, HAUS6, PENK and TXNDC4.ConclusionsMechanisms of immune defense suggested by some of the identified genes exhibit biological plausibility and may suggest novel pathways involved in the host containment of infection with TB.

Autophagy modulates the Mycobacterium tuberculosis‐induced cytokine response

Both autophagy and pro‐inflammatory cytokines are involved in the host defence against mycobacteria, but little is known regarding the effect of autophagy on Mycobacterium tuberculosis (MTB)‐induced cytokine production. In the present study, we assessed the effect of autophagy on production of monocyte‐derived and T‐cell‐derived cytokines, and examined whether two functional polymorphisms in autophagy genes led to altered cytokine production. Blocking autophagy inhibited tumour necrosis factor‐α (TNF‐α) production, while enhancing interleukin‐1β (IL‐1β) production in peripheral blood mononuclear cells stimulated with MTB. Induction of autophagy by starvation or interferon‐γ (IFN‐γ) had the opposite effect. The modulation of both TNF‐α and IL‐1β production by autophagy was induced at the level of gene transcription. Functional polymorphisms in the autophagy genes ATG16L1 and IRGM did not have a major impact on MTB‐induced cytokine production in healthy volunteers, although a moderate effect was observed on IFN‐γ production by the ATG16L1 T300A polymorphism. These data demonstrate the interplay between autophagy and inflammation during host defence against mycobacteria, and future studies to investigate the clinical implications of these effects for the susceptibility to tuberculosis are warranted.

The role of autophagy in host defence against Mycobacterium tuberculosis infection

Autophagy is a vital homeostatic process triggered by starvation and other cellular stresses, in which cytoplasmatic cargo is targeted for degradation in specialized structures termed autophagosomes. Autophagy is involved in nutrient regeneration, protein and organelle degradation, but also in clearance of intracellular pathogens such as Mycobacterium tuberculosis, the causative agent of tuberculosis. Recent studies suggest that induction of autophagy in macrophages is an effective mechanism to enhance intracellular killing of M. tuberculosis, and that the ability of the pathogen to inhibit this process is of paramount importance for its survival. Patient studies have shown genetic associations between tuberculosis and the autophagy gene IRGM, as well as with several genes indirectly involved in autophagy. In this review we will discuss the complex interplay between M. tuberculosis and autophagy, as well as the effect of polymorphisms in autophagy-related genes on susceptibility to tuberculosis.

Polymorphisms in autophagy genes and susceptibility to tuberculosis.

Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to tuberculosis (TB) among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana. We compared 22 polymorphisms of 14 autophagy genes between 1022 Indonesian TB patients and 952 matched controls, and between patients infected with different M. tuberculosis genotypes, as determined by spoligotyping. The same autophagy polymorphisms were studied in correlation with ex-vivo production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 in healthy volunteers. No association was found between TB and polymorphisms in the genes ATG10, ATG16L2, ATG2B, ATG5, ATG9B, IRGM, LAMP1, LAMP3, P2RX7, WIPI1, MTOR and ATG4C. Associations were found between polymorphisms in LAMP1 (pu200a=u200a0.02) and MTOR (pu200a=u200a0.02) and infection with the successful M. tuberculosis Beijing genotype. The polymorphisms examined were not associated with M. tuberculosis induced cytokines, except for a polymorphism in ATG10, which was linked with IL-8 production (pu200a=u200a0.04). All associations found lost statistical significance after correction for multiple testing. This first examination of a broad set of polymorphisms in autophagy genes fails to show a clear association with TB, with M. tuberculosis Beijing genotype infection or with ex-vivo pro-inflammatory cytokine production.

SNP/RD Typing of Mycobacterium tuberculosis Beijing Strains Reveals Local and Worldwide Disseminated Clonal Complexes.

The Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains.