Identification of mycobacteria to the species level by automated restriction enzyme fragment length polymorphism analysis

An automated method for the restriction fragment length polymorphism (RFLP) analysis for the differentiation of mycobacteria to the species level is described. After polymerase chain reaction (PCR) amplification of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. For accurate determination of fragment sizes the asymmetrically fluorescein-labelled PCR product was partially digested with restriction site enzymes BstEII and HaeIII. The fragments obtained were analysed electrophoretically using an automated laser fluorescence DNA sequencer. Determination of fragment sizes revealed a deviation of ±1 base pair (bp; 0.6%) when compared to expected sizes. The validity of this approach was confirmed by analysing mycobacterial DNA obtained from pure cultures of Mycobacterium (M.) tuberculosis and alcohol-fixed smears as well as paraffin-embedded sputa of patients with culture-proven tuberculosis. Additionally a diagnostic algorithm was established by investigation of cultured M. bovis, M. bovis bacille Calmette-Guérin, M. avium, M. intracellulare and M. fortuitum. The method allows the identification of restriction enzyme sites which are only 40 bp apart. Partial restriction enzyme digestion of asymmetrically fluorescence-labelled PCR products will presumably lead to the discovery of new restriction enzyme sites.     

Linfadenitis por micobacterias no tuberculosas: experiencia de 15 años

Objective

To study the epidemiology, clinical features, diagnosis, therapeutic management, and outcome of non-tuberculous mycobacterial lymphadenitis in a paediatric population of Aragón (Spain).

结核感染中IL-23R mRNA表达水平对Th17/IL-17免疫应答的影响

目的 分析白介素23受体（interleukin 23 receptor,IL-23R）对结核感染中辅助型T细胞17（T helper cell 17,Th17）免疫应答的影响,探讨IL-23R在肺结核（tuberculosis,TB）发病机制中的作用。方法 选取2015年5~10月于北京胸科医院住院的21名活动性肺结核病人（active tuberculosis,ATB）,2015年5~7月于北京昌平区结防所结核病体检的21名结核潜伏感染者（latent tuberculosis infection,LTBI）和21名健康对照（healthy donor,HD）为研究对象;分离外周血单个核细胞（peripheral blood mononuclear cell,PBMC）,并做培养;检测PBMCs中IL-23R mRNA表达水平和PBMCs上清中IL-23和IL-17A水平;比较各组IL-23R mRNA表达水平,分析IL-23R表达水平对IL-17A水平的影响。结果 IL-23R mRNA表达水平:ATB组低于LTBI组（Z=-2.528,P=0.011）,ATB组高于HD组（Z=-3.849,P<0.001）;IL-17A水平:ATB组低于LTBI组（t=2.238,P=0.031）,ATB组高于HD组（t=4.733,P<0.001）;IL-23水平在三组中的差异无统计学意义（F=0.432,P=0.651）;在ATB患者中,IL-23R mRNA表达水平与IL-17A水平呈正相关（r_s=0.438,P=0.047）。结论 结核分支杆菌感染中,IL-23R的表达水平对Th17细胞介导的免疫应答起到调节作用,可能影响到TB的易感性和感染转归。     

Infection of total hip prosthesis by <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and <Emphasis Type="Italic">Mycobacterium chelonae</Emphasis> in a patient with rheumatoid arthritis

Atypical mycobacterial infections of the musculoskeletal system are very rare and are generally associated with predisposing factors, such as trauma, use of corticosteroids, or an immunocompromised state. There have only been three reports of Mycobacterium chelonae prosthetic infection of which two cases were associated with total hip arthroplasty and one with total knee arthroplasty and no reports of both Mycobacterium tuberculosis and M. chelonae occurring in the same joint. We report a case of a patient with rheumatoid arthritis treated with low-dose methotrexate (15 mg/week) who developed infection with both M. tuberculosis and M. chelonae after the revision of a prosthetic hip. Joint infections by mycobacteria are clinically indistinguishable from those caused by more common bacterial pathogens and, therefore, diagnosis is often delayed. Recurrent prosthetic hip infections, particularly in immunosuppressive patients, should alert the physician to consider the possibility of both tuberculous and atypical mycobacterial infections. Obtaining appropriate cultures can be critical in making the diagnosis and directing treatment. With the increasing use of immunosuppressive agents, including TNF alpha inhibitors, it is likely that there will be an increase in the number of mycobacterial infections complicating arthroplasties.     

IP-10 contributes to the inhibition of mycobacterial growth in an ex vivo whole blood assay

Interferon-γ inducible protein 10 (IP-10), is a potent chemoattractant that promotes migration of monocytes and activated T-cells to inflammation foci. IP-10 is elevated in serum of patients with chronic hepatitis C virus (HCV) and tuberculosis (TB) infections, although it remains to be determined the contribution of IP-10 in restricting Mycobacterium tuberculosis (Mtb) replication. Here, we investigated the impact of IP-10 on mycobacteria replication using the ex vivo model of human whole-blood (WB) assay. In particular, we compared the levels of IP-10 upon infection with different Mtb clinical strains and species of non-tuberculous mycobacteria (NTM) and evaluated how IP-10 may contain bacterial replication. Interestingly, we observed that the inhibition of the host enzyme dipeptidyl peptidase IV (DPP-IV), which inactivates IP-10 through cleavage of two amino acids at the chemokine N-terminus, restricted mycobacterial persistence in WB, supporting the critical role of full length IP-10 in mediating an anti-Mtb response. Addition of recombinant IP-10 expressed in eukaryotic cells enhanced the anti-mycobacterial activity in WB, although no differences were observed when IP-10 containing different proportions of cleaved and non-cleaved forms of the chemokine were added. Moreover, recombinant IP-10 did not exert a direct anti-mycobacterial effect. Our results underscore the clinical relevance of IP-10 in mycobacteria pathogenesis and support the potential outcomes that may derive by targeting the IP-10/CXCR3 pathway as host directed therapies for the treatment of Mtb or NTM infections.     

溶血离心培养法检测肺结核患者外周血分支杆菌及其L型

目的　建立一种自血液中分离分支杆菌及其L型的新方法。方法　肺结核患者的外周血直接或溶血离心后取沉淀种入 92 3TB和 92 3TBL液体培养基培养 ,培养物用免疫酶染色 (ABC法 )鉴定。结果　 6 5份标本分支杆菌和分支杆菌L型血培养的阳性率、总检出率分别为 15 %、2 6 %、32 % ;溶血离心培养法的阳性率明显高于直接培养法 (P <0 0 5 ) ;相应痰培养中 ,抗酸杆菌和抗酸菌L型的阳性率、总检出率分别为 38%、2 0 %和 5 2 % ,高于肺结核患者外周血培养的阳性率 (P <0 0 5 ) ;但血、痰配对检测总阳性率可达 6 5 %。结论　肺结核患者外周血存在分支杆菌及其L型 ,并以L型为主 ;溶血离心液体培养结合免疫酶染色可常规用于检测血液中分支杆菌及其L型 ;结合痰标本检测 ,可提高肺结核患者细菌学检查的阳性率     

痰标本PCR-反向斑点杂交法对疑似非结核分枝杆菌肺病的诊断价值

目的 探讨痰PCR-反向斑点杂交法对疑似非结核分枝杆菌(NTM)肺病的诊断价值。方法 搜集2014年1月至2017年10月同济大学附属上海市肺科医院收治的、通过临床症状和影像学检查疑诊为NTM肺病的患者334例,分别取痰液行PCR-反向斑点杂交法分枝杆菌菌种鉴定基因检测及分枝杆菌传统培养法[对硝基苯甲酸(PNB)、噻吩-2-羧酸肼(TCH)培养基生长试验]检测,最终确诊为NTM肺病患者218例(NTM肺病组)、结核病患者42例(结核病组)、其他肺部疾病患者74例(研究中予以排除)。以最终确诊结果为金标准,对痰PCR-反向斑点杂交法诊断临床疑诊NTM肺病患者的敏感度、特异度、阳性预测值和符合率进行分析。结果 疑诊NTM肺病的334例患者中,培养法分枝杆菌检出率为67.66%(226/334),PCR-反向斑点杂交法分枝杆菌检出率为64.67%(216/334),两种方法分枝杆菌检出率的比较,差异无统计学意义(χ ²=0.67,P=0.231)。确诊的218例NTM肺病患者中,PCR-反向斑点杂交法检出NTM的敏感度为82.57%(180/218),阳性预测值为98.36%(180/183);培养法检出NTM的敏感度为87.61%(191/218),阳性预测值为100.00%(191/191);两种方法检出NTM的敏感度比较,差异无统计学意义(χ ²=1.20,P=0.169)。以42例结核病组患者作为对照,PCR-反向斑点杂交法检测的特异度为78.57%(33/42),诊断结核病的阳性预测值为100.00%(33/33);培养法的特异度为83.33%(35/42),诊断结核病的阳性预测值为100.00%(35/35);两种检测方法特异度的比较,差异无统计学意义(χ ²=0.31,P=0.391)。PCR-反向斑点杂交法在两组患者中的诊断符合率为83.08%(216/260),培养法在两组患者中的诊断符合率为86.92%(226/260),差异无统计学意义(χ ²=1.51,P=0.134)。 结论 以胸部CT检查为诊断基础的疑似NTM肺病患者中,应用PCR-反向斑点杂交法行痰液NTM检测具有较高的敏感度和特异度,对临床的快速准确诊断和及时治疗有一定的参考意义。     

结核分枝杆菌免疫逃逸机制的研究进展

结核分枝杆菌是一种烈性、强毒、传染性病原菌。由其引发的结核病极难治愈,普通的抗生素药物对其根本不起作用。究其原因是该菌通过一系列目前仍尚未阐明的逃逸机制逃脱了巨噬细胞溶酶体对其的融合杀伤作用,因而得以存活并寄生在巨噬细胞内的吞噬体中。对此,近年来发表了一系列极有价值的研究成果,主要集中在可溶性N-乙基顺丁希二酰亚胺敏感因子连接蛋白受体（SNARE）融合核心复合体的形成,Rab7、Rab5及其效应蛋白EEA-1在晚内体成熟中的作用以及自噬在免疫逃逸机制中的作用等几个方面,这对于进一步丰富和阐明结核分枝杆菌的免疫逃逸机制理论具有重要意义。     

Development of vaccines against tuberculosis

Development of an effective vaccine against tuberculosis (TB) hinges on an improved understanding of the human immune responses to Mycobacterium tuberculosis. A successful vaccination strategy should be able to stimulate the appropriate arm of the immune system with concomitant generation of the memory cells. In the absence of a perfect strategy, while long term efforts of TB researchers continue to resolve the nature of protective immunity against TB and other related issues, the current approach, dictated by the urgency of a TB vaccine, employs available knowledge and technology to develop new TB vaccines and channel the promising ones to clinical trials. While Indian scientists have contributed in several areas towards the development of a TB vaccine, this review is an attempt to summarize their contributions mainly pertaining to the discovery of new antigens, immune responses elicited by antigens against TB and development of new vaccines and their evaluation in animal models.     

Genetic control of immune-mediated necrosis of Mycobacterium avium granulomas

Intravenous infection of C57BL/6 and C57BL/10 mice with low doses of a highly virulent strain of Mycobacterium avium (ATCC 25291) led to the development of granulomas that underwent necrosis. In contrast, neither BALB/c nor DBA/1 mice developed granuloma necrosis after such infection despite a similar course of mycobacterial proliferation. Studies with C57BL/10 mice congenic for the Hc locus revealed that an intact complement C5 gene is required for granuloma necrosis. On the other hand, genetic disruption of the interleukin-10 gene in BALB/c mice made this strain susceptible to granuloma necrosis.