Tuberculosis vaccine design: influence of the completed genome sequence

Tuberculosis continues to be a major health problem, with more adults dying from Mycobacterium tuberculosis than any other pathogen world-wide.With the onset of the HIV epidemic and an increase in drug-resistant M. tuberculosis strains, the need for an improved vaccine has become an international priority.The recent completion of the genome sequences for two M. tuberculosis strains provides a wealth of information that can be used to design new strategies for vaccine development. The challenge comes in making rational choices from among the 4,000 genes of the most probable candidate immunogens or virulence genes.Thus, a well-designed screen is needed to reduce the number of candidates that must be tested. Presently, the most valuable role that bioinformatics can play is to provide such a screen.     

Extensively drug-resistant tuberculosis: current challenges and threats

Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis caused by a Mycobacterium tuberculosis strain that is resistant to at least rifampicin and isoniazid among the first-line antitubercular drugs (multidrug-resistant tuberculosis; MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second-line drugs, namely amikacin, kanamycin and/or capreomycin. Recent studies have described XDR-TB strains from all continents. Worldwide prevalence of XDR-TB is estimated to be c. 6.6% in all the studied countries among multidrug-resistant M. tuberculosis strains. The emergence of XDR-TB strains is a reflection of poor tuberculosis management, and controlling its emergence constitutes an urgent global health reality and a challenge to tuberculosis control activities in all parts of the world, especially in developing countries and those lacking resources and as well as in countries with increasing prevalence of HIV/AIDS.     

结核分枝杆菌耐药性的分子生物学研究进展

结核病一直是世界性问题,我国其发病情况尤为严重,是亚洲的第二大结核病发病国家。结核病治疗方面常使用抗生素作为首选药物,随着抗菌药的滥用,结核杆菌对多种抗菌药产生耐药性,结核病耐药患者增多,治疗难度增加。因此,结核杆菌耐药分子机制的研究更加重要,新型抗结核药物研制更加迫切。结核分枝杆菌的基因突变是引起耐药的主要分子学依据,因此基于结核分枝杆菌耐药性相关基因的深入探索,对于预防结核病的传播及治疗皆具有深远影响。本文从分子生物学角度分析了近年来结核分枝杆菌耐药性产生的原因及相关研究进展。     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

Occupational risk of tuberculosis transmission in a low incidence area

Background

To investigate the occupational risk of tuberculosis (TB) infection in a low-incidence setting, data from a prospective study of patients with culture-confirmed TB conducted in Hamburg, Germany, from 1997 to 2002 were evaluated.

Methods

M. tuberculosis isolates were genotyped by IS6110 RFLP analysis. Results of contact tracing and additional patient interviews were used for further epidemiological analyses.

Results

Out of 848 cases included in the cluster analysis, 286 (33.7%) were classified into 76 clusters comprising 2 to 39 patients. In total, two patients in the non-cluster and eight patients in the cluster group were health-care workers. Logistic regression analysis confirmed work in the health-care sector as the strongest predictor for clustering (OR 17.9). However, only two of the eight transmission links among the eight clusters involving health-care workers had been detected previously. Overall, conventional contact tracing performed before genotyping had identified only 26 (25.2%) of the 103 contact persons with the disease among the clustered cases whose transmission links were epidemiologically verified.

Conclusion

Recent transmission was found to be strongly associated with health-care work in a setting with low incidence of TB. Conventional contact tracing alone was shown to be insufficient to discover recent transmission chains. The data presented also indicate the need for establishing improved TB control strategies in health-care settings.     

结核分枝杆菌潜伏感染诊断方法的新进展

每年有超过8百万人感染结核,其中绝大部分没有发展为活动性结核病,而是表现为潜伏性结核感染。大多数活动性结核病是潜伏感染的结核杆菌重新被激活所致,因此结核潜伏感染者成为结核患者的重要来源。及早诊断和治疗结核潜伏感染者是控制结核传播的最有效手段之一。我们较要综述了目前国内外结核潜伏感染的诊断方法及其新进展。     

Immunoreactivity of five antigens of Mycobacterium tuberculosis in patients attending a public health care facility in an area with high endemicity for TB

The objective of this study was to evaluate people attending a primary health clinic in Rio de Janeiro, Brazil for immunoreactivity to five Mycobacterium tuberculosis antigens, as these antigens are markers of immune response and factors associated with active TB. The serum antibody titers of different categories of patients (defined by microbiological and radiological characteristics and by response to therapy on follow‐up) to 38 kDa, 16 kDa, MPT64, ESAT‐6 and MT10.3 antigens were determined blind with ELISA. Positive tests to each antigen were defined with ROC analysis. OR were calculated for factors associated with humoral response in patients with active TB. A total of 201 patients underwent serological testing. Patients with confirmed active TB responded more frequently to MPT64 (44%), 16 kDa (37.7%) and 38 kDa (36.1%). ESAT‐6 and MT10.3 were also able to distinguish people in TB groups from controls. TB infected subjects responded less frequently to ESAT‐6 and MT10.3 (3.7% and 11%, respectively). Sensitivity and specificity to all antigens combined were 58.4% and 60.7%, respectively. Reactivity to 38 kDa and to MPT64 was more likely among alcohol users OR 2.61 (95%CI;1.05–6.94) and OR 3.27 (95%CI;1.33–8.15), respectively. 16 kDa antigen elicited a more protective response among smokers, OR 0.29 (95%CI; 0.10–0.83). It was concluded that reactivity to all antigens tested represented markers of active disease. ESAT‐6 and MT10.3 could not be identified as markers of TB infection in this community. Sensitivity was higher to all antigens combined, but at a cost of lower specificity. Interestingly, among factors associated with positive immunoreactivity, alcohol use and smoking seem to polarize the humoral response in different directions. This finding deserves further investigation.     

结核分枝杆菌抗原检测研究进展

结核病仍然是一个严重的全球性公共卫生问题,有效控制结核病的障碍在于缺乏早期、准确的诊断方法。机体受到结核分枝杆菌感染后,体内首先出现的是结核分枝杆菌特异性抗原。因此,结核分枝杆菌抗原检测作为结核病早期诊断的方法可能具有很高的诊断价值。我们简要综述了结核分枝杆菌抗原检测的相关研究进展。     

Horizontal transfer of a virulence operon to the ancestor of Mycobacterium tuberculosis

The contribution of interspecies horizontal gene transfer (HGT) to the evolution and virulence of Mycobacterium tuberculosis, the agent of tuberculosis in humans, has been barely investigated. Here we have studied the evolutionary history of the M. tuberculosis Rv0986-8 virulence operon recently identified, through functional genomics approaches, as playing an important role in parasitism of host phagocytic cells. We showed that among actinobacteria, this operon is specific to the M. tuberculosis complex and to ancestral Mycobacterium prototuberculosis species. These data, together with phylogenetic reconstruction and other in silico analyses, provided strong evidence that this operon has been acquired horizontally by the ancestor of M. tuberculosis, before the recent evolutionary bottleneck that preceded the clonal-like evolution of the M. tuberculosis complex. Genomic signature profiling further suggested that the transfer was plasmid mediated and that the operon originated from a gamma-proteobacterium donor species. Our study points out for the first time the contribution of HGT to the emergence of M. tuberculosis and close relatives as major pathogens. In addition, our data underline the importance of deciphering gene transfer networks in M. tuberculosis in order to better understand the evolutionary mechanisms involved in mycobacterial virulence.     

Structural implications of lipoarabinomannan glycans from global clinical isolates in diagnosis of Mycobacterium tuberculosis infection

In Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure–function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates (HN878, EAI, and IO) and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAM-methylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal β-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has ∼50% of α−arabinofuranose -(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM.     

结核潜伏感染动物模型的研究进展

结核潜伏感染(latent tuberculosis infection, LTBI)是机体对结核分枝杆菌抗原持续性免疫反应的状态,既无活动性结核病临床症状,也无结核病影像学表现。LTBI激活是新发结核病的主要来源之一。LTBI动物模型的建立是研究结核的潜伏感染和复发机制,开发诊断试剂,评估抗结核新药、新疫苗的有效性、安全性的基础。建立稳定、成本低、易推广、潜伏期时长适中、复发起点和复发水平变异小的LTBI动物模型,是其未来研究发展的方向。本文就LTBI动物模型的研究进展进行综述,期望为结核病防治工作者提供参考资料。     

Recent updates on drug resistance in Mycobacterium tuberculosis

Tuberculosis (TB) along with acquired immune deficiency syndrome and malaria rank among the top three fatal infectious diseases which pose threat to global public health, especially in middle and low income countries. TB caused by Mycobacterium tuberculosis (Mtb) is an airborne infectious disease and one-third of the world's population gets infected with TB leading to nearly 1·6 million deaths annually. TB drugs are administered in different combinations of four first-line drugs (rifampicin, isoniazid, pyrazinamide and ethambutol) which form the core of treatment regimens in the initial treatment phase of 6–9 months. Several reasons account for the failure of TB therapy such as (i) late diagnosis, (ii) lack of timely and proper administration of effective drugs, (iii) lower availability of less toxic, inexpensive and effective drugs, (iv) long treatment duration, (v) nonadherence to drug regimen and (vi) evolution of drug-resistant TB strains. Drug-resistant TB poses a significant challenge to TB therapy and control programs. In the background of worldwide emergence of 558 000 new TB cases with resistance to rifampicin in the year 2017 and of them, 82% becoming multidrug-resistant TB (MDR-TB), it is essential to continuously update the knowledge on the mechanisms and molecular basis for evolution of Mtb drug resistance. This narrative and traditional review summarizes the progress on the anti-tubercular agents, their mode of action and drug resistance mechanisms in Mtb. The aim of this review is to provide recent updates on drug resistance mechanisms, newly developed/repurposed anti-TB agents in pipeline and international recommendations to manage MDR-TB. It is based on recent literature and WHO guidelines and aims to facilitate better understanding of drug resistance for effective TB therapy and clinical management.     

非一线抗结核药物耐药机制及耐药性诊断研究进展

结核病(Tuberculosis, TB)至今仍是世界三大传染疾病之一。2014年,TB导致的死亡人数已经超过HIV。二线抗TB药物是临床治疗耐多药TB(Multidrug-resistant TB, MDR-TB)的主要药物,然而某些MDR-TB患者由于未及时诊断、治疗方案不合理、所处区域医疗条件差等原因,逐渐发展成为广泛耐药TB(Extensively drug-resistant TB, XDR-TB),使治疗更加困难,其死亡率甚至与肺癌接近。目前结核分枝杆菌(Mycobacterium tuberculosis)的耐药性机制研究已经转向非一线药物,如二线、三线和一些新研发的抗TB药物,揭示这些非一线药物的耐药机制对于耐药TB的治疗和新型抗TB药物的研发具有重要意义。本文对目前临床上使用的主要非一线药物的耐药机制研究进行了综述,并对目前常用的TB耐药性诊断方法的优缺点进行了归纳比较。     

A population-based study of first and second-line drug-resistant tuberculosis in a high-burden area of the Mexico/United States border

The resistance of 139 Mycobacterium tuberculosis (MTB) isolates from the city of Monterrey, Northeast Mexico, to first and second-line anti-TB drugs was analysed. A total of 73 isolates were susceptible and 66 were resistant to anti-TB drugs. Monoresistance to streptomycin, isoniazid (INH) and ethambutol was observed in 29 cases. Resistance to INH was found in 52 cases and in 29 cases INH resistance was combined with resistance to two or three drugs. A total of 24 isolates were multidrug-resistant (MDR) resistant to at least INH and rifampicin and 11 MDR cases were resistant to five drugs. The proportion of MDR-TB among new TB cases in our target population was 0.72% (1/139 cases). The proportion of MDR-TB among previously treated cases was 25.18% (35/139 cases). The 13 polyresistant and 24 MDR isolates were assayed against the following seven second-line drugs: amikacin (AMK), kanamycin (KAN), capreomycin (CAP), clofazimine (CLF), ethionamide (ETH), ofloxacin (OFL) and cycloserine (CLS). Resistance to CLF, OFL or CLS was not observed. Resistance was detected to ETH (10.80%) and to AMK (2.70%), KAN (2.70%) and CAP (2.70%). One isolate of MDR with primary resistance was also resistant to three second-line drugs. Monterrey has a high prevalence of MDR-TB among previously treated cases and extensively drug-resistant-MTB strains may soon appear.     

Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis

The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. Proteins 2015; 83:389–394. © 2014 Wiley Periodicals, Inc.     

HIV合并肺结核与单纯肺结核的临床特征分析及抗结核治疗效果对比

目的:分析HIV合并肺结核患者的临床特征及抗结核治疗的疗效。方法:将我院收治的HIV感染合并肺结核初治患者53例作为A组,将同期收治的单纯肺结核患者176例作为B组,对两组患者临床资料、实验室检查结果以及治疗效果等进行回顾性分析。结果:A组患者并发症发生率以及肺外结核发生率显著高于B组(P0.05),A组咳嗽发生率低于B组,但发热发生率高于B组(P0.05);A组患者斑点实验、PPD实验、痰查抗酸杆菌阳性率均低于B组(P0.05),A组患者肝功能异常、肾功能异常以及CD4+计数200发生率高于B组(P0.05);A组抗结核治疗的临床疗效低于B组(P0.05);两组患者治疗后CD4+水平均高于治疗前,且B组高于A组(P0.05)。结论:与单纯感染肺结核的患者相比,HIV合并肺结核患者并发症以及肺外结核发生率较高,实验室相关检查敏感性较低,抗结核治疗的效果较差,临床应给予重视。     

结核分枝杆菌的致病机理

结核病仍是全球健康的主要威胁,其致病菌结核分枝杆菌的致闰机理与众不同。脂类代谢在致病中具有重要的作用。巨噬细胞被入侵的细菌修的机理也是研究细菌持续感染致病的重要突破点,研究细菌的致病机理可以为开发新的疫苗和治疗药物奠定坚实的基础。     

全球基金结核病项目对东莞市流动人口结核病防治效果分析

目的评价全球基金对流动人口结核病防治项目工作中的作用,为探索流动人口结核病防治策略提供科学依据。方法收集、比较分析项目实施前后东莞市流动人口肺结核患者发现情况。结果实施全球基金项目后,东莞市流动人口肺结核可疑患者发现人数及确诊病例数明显增高。结论全球基金项目对东莞市流动人口肺结核患者的发现率发挥积极作用,为制定科学的流动人口结核病防治策略打下坚实基础。     

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.