Live attenuated mutants of Mycobacterium tuberculosis as candidate vaccines against tuberculosis

The recent advances in genetic tools to manipulate Mycobacterium tuberculosis have led to the construction of defined mutants and to the study of their role in the virulence and pathogenesis of tuberculosis. The safety and vaccine potential of a few of these M. tuberculosis mutants as candidate vaccines against tuberculosis are discussed.     

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。     

卡波姆基质帕司烟肼凝胶体外抗结核活性及动物实验的研究

目的　观察卡波姆凝胶与帕司烟肼联合体外抗结核作用和支气管介入的安全性。方法　手工法、仪器法分别测定卡波姆凝胶与帕司烟肼药物的最小抑菌浓度和最小杀菌浓度及家兔经支气管介入的安全性试验。结果　帕司烟肼的卡波姆凝胶对H37Rv标准株、牛型结核分枝杆菌、草分枝杆菌MIC值分别为 0 1、0 1、0 4mg/L ,MBC值分别为 0 2、0 2和 1 6mg/L ;帕司烟肼的卡波姆凝胶与帕司烟肼单体MIC、MBC值无显著差异 ;家兔动物实验表明该药无任何毒副作用。结论　帕司烟肼凝胶具有与帕司烟肼单体相同的抗结核菌药效 ,卡波姆基质不影响帕司烟肼的抗菌活性 ;以卡波姆为基质的帕司烟肼凝胶应用安全     

In vivo persistence and protective efficacy of the bacille Calmette Guerin vaccine overexpressing the HspX latency antigen

New strategies to control infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, are urgently required, particularly in areas where acquired immunodeficiencies are prevalent. In this report we have determined if modification of the current tuberculosis vaccine, Mycobacterium bovis BCG, to constitutively express the mycobacterial HspX latency antigen altered its protective effect against challenge with virulent M. tuberculosis. Overexpression of M. tuberculosis HspX in BCG caused reduced growth in aerated cultures compared to control BCG, but growth under limited oxygen availability was not markedly altered. Upon infection of mice, BCG:HspX displayed tissue-specific attenuation compared to control BCG, with reduced growth within the lung and liver but not the spleen. Both BCG:HspX and control BCG protected mice against aerosol M. tuberculosis challenge to a similar extent, however, immunodeficient mice infected with BCG:HspX survived significantly longer than mice infected with the control BCG strain. Therefore, altering the in vivo persistence of BCG by overexpression of HspX may be one important step towards developing a new tuberculosis vaccine with an improved safety profile and suitable protective efficacy against M. tuberculosis infection.     

候选结核疫苗的评价模型

卡介苗作为目前唯一批准使用的预防结核疫苗,由于无法提供充足的保护力,目前结核病依然是严重危害人类健康的传染病,因此筛选优质候选疫苗迫在眉睫。所有候选疫苗进入临床试验前必须评价其安全性、免疫原性以及有效性。评价结果在一定程度上也预示候选疫苗在人体的免疫作用,也是目前评价候选疫苗是否优于传统卡介苗的依据。论述候选结核疫苗临床试验前所使用的体内和体外评价模型。对评价模型的充分认识将提高候选疫苗评价数据对人体临床试验的预测意义,也可更具体地执行实验动物的替代、减少和优化原则。     

Lysine auxotrophy combined with deletion of the SecA2 gene results in a safe and highly immunogenic candidate live attenuated vaccine for tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major global health problem, despite the widespread use of the M. bovis Bacille Calmette-Guerin (BCG) vaccine and the availability of drug therapies. In recent years, the high incidence of coinfection of M. tuberculosis and HIV, as well as escalating problems associated with drug resistance, has raised ominous concerns with regard to TB control. Vaccination with BCG has not proven highly effective in controlling TB, and also has been associated with increasing concerns about the potential for the vaccine to cause disseminated mycobacterial infection in HIV infected hosts. Thus, the development of an efficacious and safe TB vaccine is generally viewed as a critical to achieving control of the ongoing global TB pandemic. In the current study, we have analyzed the vaccine efficacy of an attenuated M. tuberculosis strain that combines a mutation that enhances T cell priming (ΔsecA2) with a strongly attenuating lysine auxotrophy mutation (ΔlysA). The ΔsecA2 mutant was previously shown to be defective in the inhibition of apoptosis and markedly increased priming of antigen-specific CD8(+) T cells in vivo. Similarly, the ΔsecA2ΔlysA strain retained enhanced apoptosis and augmented CD8(+) T cell stimulatory effects, but with a noticeably improved safety profile in immunosuppressed mice. Thus, the M. tuberculosis ΔsecA2ΔlysA mutant represents a live attenuated TB vaccine strain with the potential to deliver increased protection and safety compared to standard BCG vaccination.     

The Ethics of Isolation for Patients With Tuberculosis in Australia

This case study examines the ethical dimensions of isolation for patients diagnosed with tuberculosis (TB) in Australia. It seeks to explore the issues of resource allocation, liberty, and public safety for wider consideration and discussion.     

A nose-only apparatus for airborne delivery of Mycobacterium tuberculosis to mice: calibration of biological parameters

Mycobacterium tuberculosis is the main cause of tuberculosis and is still a public health concern worldwide. This mycobacterium is transmitted through aerosols from human beings suffering from pulmonary tuberculosis to susceptible persons. To study this natural route of infection, we designed a new nose-only aerosol apparatus--system of aerosolisation of microorganisms (SAM)--in a carefully designed biohazard facility. For safety reasons, Mycobacterium smegmatis was first used to calibrate several parameters, such as inoculum density, atmospheric conditions (i.e. hygrometry) and particle size distribution. We present evidence that our apparatus is totally adapted to airborne delivery; the particle size of generated aerosol ranges from 1 to 7 microm, which is ideal for an infection by inhalation. We found that 99% of generated particles (<7 microm) could be retained by the respiratory tract, and among these particles, 62-79% (<3.3 microm) were able to reach pulmonary compartments. The next step was to simultaneously challenge 48 mice with M. tuberculosis in a highly reproducible way. We showed that a moderate dose (4 log10 colony-forming units (CFU) per mice) of M. tuberculosis was capable of causing progressive lung pathology and death in mice 30 days post-aerosolisation. Therefore, our apparatus, once calibrated, is easy to handle, safe, and can be used with any pathogen, which is spread by aerosol.     

Safety of Resuming Tumor Necrosis Factor Inhibitors in Ankylosing Spondylitis Patients Concomitant with the Treatment of Active Tuberculosis: A Retrospective Nationwide Registry of the Korean Society of Spondyloarthritis Research

BackgroundsPatients who develop an active tuberculosis infection during tumor necrosis factor (TNF) inhibitor treatment typically discontinue TNF inhibitor and receive standard anti-tuberculosis treatment. However, there is currently insufficient information on patient outcomes following resumption of TNF inhibitor treatment during ongoing anti- tuberculosis treatment. Our study was designed to investigate the safety of resuming TNF inhibitors in ankylosing spondylitis (AS) patients who developed tuberculosis as a complication of the use of TNF inhibitors.MethodsThrough the nationwide registry of the Korean Society of Spondyloarthritis Research, 3929 AS patients who were prescribed TNF inhibitors were recruited between June 2003 and June 2014 at fourteen referral hospitals. Clinical information was analyzed about the patients who experienced tuberculosis after exposure to TNF inhibitors. The clinical features of resumers and non-resumers of TNF inhibitors were compared and the outcomes of tuberculosis were surveyed individually.FindingsFifty-six AS patients were treated for tuberculosis associated with TNF inhibitors. Among them, 23 patients resumed TNF inhibitors, and these patients were found to be exposed to TNF inhibitors for a longer period of time and experienced more frequent disease flare-up after discontinuation of TNF inhibitors compared with those who did not resume. Fifteen patients resumed TNF inhibitors during anti-tuberculosis treatment (early resumers) and 8 after completion of anti-tuberculosis treatment (late resumers). Median time to resuming TNF inhibitor from tuberculosis was 3.3 and 9.0 months in the early and late resumers, respectively. Tuberculosis was treated successfully in all resumers and did not relapse in any of them during follow-up (median 33.8 [IQR; 20.8–66.7] months).ConclusionsInstances of tuberculosis were treated successfully in our AS patients, even when given concomitantly with TNF inhibitors. We suggest that early resumption of TNF inhibitors in AS patients could be safe under effective coverage of tuberculosis.     

Pulmonary paragonimiasis in Southeast Asians living in the central San Joaquin Valley.

We describe four cases of pulmonary paragonimiasis in Southeast Asians who emigrated to the central San Joaquin Valley of California. Physicians should be alerted to the possibility of this disease in Asian patients with hemoptysis and pulmonary infiltrates. Paragonimiasis can masquerade as pulmonary tuberculosis, especially in patients from areas that are endemic for both the parasite and the tubercle bacillus. The ease and safety with which this infection can be treated, in contrast to tuberculosis, reiterates the importance of diagnosing this lung fluke when it is present.     

Effects of chromium on the immune system

The performance of integral membrane antigens (IMAs) of Mycobacterium habana TMC 5135 in detecting antimycobacterial antibodies in serum and body fluids of patients mainly of extrapulmonary tuberculosis was evaluated. The IMAs were recovered from the detergent phase during Triton X-114 treatment of the plasma membrane of M. habana. Antimycobacterial antibodies were detected by ELISA using IMAs in serum and body fluids of 42 patients and 62 control subjects. As authentic adjunct Mycobacterium tuberculosis antigens were also detected (by ELISA) in body fluids and circulating immune complexes using anti-M. tuberculosis H37Ra antibodies. Anti-M. habana IMA antibody detection increased the positivity rate from 26.% (11/42) and 10% (4/42) obtained by culture and smear microscopy, respectively, to 86% (36/42). M. tuberculosis antigens were also found in 29 out of 36 anti-M. habana IMA antibody-positive cases. Interestingly, all 11 culture-positive cases were also positive for anti-M. habana IMA antibodies. The mean antigen titres in 23 cases, positive for antigens in body fluids, were 2.34 times higher in those who were also positive for anti-IMA antibodies in serum than in those negative for these antibodies. M. habana IMAs may be promising non-tubercular candidate antigens in ELISA-based serodiagnosis of extrapulmonary tuberculosis with substantial sensitivity, specificity and safety.     

不同治疗方案治疗脊柱结核的效果对比

目的:分析不同脊柱结核手术方式在治疗效果和治疗安全性方面的差异。方法:将我院脊柱外科施以手术治疗的89例脊柱结核患者为研究对象,根据手术方法差异分成后路组45例和前路组44例,记录手术评价指标、Cobb角、手术前后美国脊髓损伤学会神经功能(ASIA评分)变化和术后并发症,并对记录结果行统计分析。结果:后路组手术时间(185.71±21.89)min、出血量(503.12±57.81)m L、出院时间(21.43±3.52)d、手术相关不良反应率(6.82%)、Cobb角度(18.34±8.41)°显著低于前路组,而后路组神经损伤治愈率(54.55%)高于前路组,P0.05,存在统计学差异。结论:手术入路能够影响脊柱结核手术的治疗效果,与前入路手术相比,后入路手术在手术简便性、安全性、有效性方面优势明显。     

Improved protection by recombinant BCG

Mycobacterium bovis bacille Calmette Guérin (BCG) is one of the most widely used live vaccines. Technologic advancement in genome manipulation enables the construction of recombinant BCG (rBCG) strains, which can be employed as highly immunogenic vaccines against tuberculosis with improved safety profile.     

Rapid and Biosecure Diagnostic Test for Tuberculosis

Early and rapid detection of the causative organism is necessary in tuberculosis. We present here an integrated and dedicated molecular biology system for tuberculosis diagnosis. One hundred and eighty-nine (189) biologic specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl–Neelsen staining, cultivation on a solid medium, and by a balanced heminested fluorometric PCR system (Orange G3TB) that preserves worker safety and produces a rather pure material free of potential inhibitors. DNA amplification was carried out in a low cost using a tuberculosis thermocycler-fluorometer. The double stranded DNA produced is fluorometrically detected. The whole reaction is carried out in one single tube which is never opened after adding the processed sample, thus minimizing the risk of cross contamination with amplicons. The assay is able to detect 30 bacilli/ml of sample having a 99.8 % inter-assay coefficient of variation. PCR was positive in 36 (18.9 %) tested samples (33 of them were smear-negative). In our study, it yields a preliminary overall sensitivity of 97.4 %. In addition, its overall specificity is 98.7 %. The total run time of the test is 4 h with two and a half real working hours. All PCR-positive samples also had a positive result by microbiological culture and clinical criteria. The results obtained showed that it could be a very useful tool to increase efficiency in detecting the tuberculosis disease in low bacillus inoculum samples. Furthermore, its low cost and friendly usage make it feasible to be used in regions with poor development.     

64层螺旋CT不同剂量扫描对活动性继发性肺结核患者诊断价值、图像质量和辐射剂量的影响

目的:研究64层螺旋CT不同剂量扫描对活动性继发性肺结核患者诊断价值、图像质量和辐射剂量的影响,旨在为临床活动性继发性肺结核患者提供更为安全有效的检查方案。方法:将我院从2018年1月2020年1月收治的100例活动性继发性肺结核患者纳入研究。对所有患者分别进行低剂量(40 m A)以及常规剂量(120 m A)扫描和薄层重建。比较不同剂量扫描的诊断准确率、影像学表现、图像质量以及辐射参数、患者满意度。结果:64层螺旋CT低剂量扫描与常规剂量扫描诊断活动性继发性肺结核的准确率分别为92.00%、93.00%,两者比较差异无统计学意义(P>0.05)。低剂量扫描及常规剂量扫描在空洞、钙化灶、实变、磨玻璃密度影及树芽征发生率方面对比差异均无统计学意义(P>0.05)。低剂量扫描及常规剂量扫描在扫描图像质量、薄层重建图像质量方面对比差异均无统计学意义(P>0.05)。低剂量扫描各项扫描辐射参数均低于常规剂量扫描,差异有统计学意义(P<0.05)。低剂量扫描的患者满意度高于常规剂量扫描的患者(P<0.05)。结论:64层螺旋CT不同剂量扫描对活动性继发性肺结核患者的诊断价值及生成的图像质量相近,但低剂量扫描可有效降低辐射剂量,从而在一定程度上增加扫描的安全性,使患者满意度提升。     

局部注药联合口服康复新液辅助治疗颈淋巴结结核疗效观察

目的:探讨局部注射药物联合口服康复新液辅助治疗颈淋巴结结核的临床疗效及安全性。方法:按照随机数字表法将2010年1月-2014年1月我院收治的140例颈淋巴结结核患者分为对照组(n=70)和观察组(n=70),对照组口服异烟肼、利福平、乙胺丁醇、吡嗪酰胺及肌注链霉素进行全身抗结核治疗,观察组在对照组的基础上局部注射异烟肼、链霉素粉联合口服康复新液治疗。比较两组患者治疗后1个月、3个月及6个月症状缓解率,淋巴结吸收率,治疗后3个月复发率及不良反应发生率。结果:观察组治疗后1个月、3个月及6个月的症状缓解率、淋巴结吸收率均高于对照组(P0.05)。治疗后3个月,观察组复发率为2.86%,显著低于对照组的11.43%(P0.05)。观察组恶心、局部反应、白细胞减少及肝功能损害发生率均低于对照组(P0.05)。结论:局部注射抗结核药物联合口服康复新液辅助治疗颈淋巴结结核可提高患者的症状缓解率和淋巴结吸收率,且安全性高,有重要的临床推广价值。     

The safety, effectiveness and concentrations of adjusted lopinavir/ritonavir in HIV-infected adults on rifampicin-based antitubercular therapy

Objective

Rifampicin co-administration dramatically reduces plasma lopinavir concentrations. Studies in healthy volunteers and HIV-infected patients showed that doubling the dose of lopinavir/ritonavir (LPV/r) or adding additional ritonavir offsets this interaction. However, high rates of hepatotoxicity were observed in healthy volunteers. We evaluated the safety, effectiveness and pre-dose concentrations of adjusted doses of LPV/r in HIV infected adults treated with rifampicin-based tuberculosis treatment.

Methods

Adult patients on a LPV/r-based antiretroviral regimen and rifampicin-based tuberculosis therapy were enrolled. Doubled doses of LPV/r or an additional 300 mg of ritonavir were used to overcome the inducing effect of rifampicin. Steady-state lopinavir pre-dose concentrations were evaluated every second month.

Results

18 patients were enrolled with a total of 79 patient months of observation. 11/18 patients were followed up until tuberculosis treatment completion. During tuberculosis treatment, the median (IQR) pre-dose lopinavir concentration was 6.8 (1.1–9.2) mg/L and 36/47 (77%) were above the recommended trough concentration of 1 mg/L. Treatment was generally well tolerated with no grade 3 or 4 toxicity: 8 patients developed grade 1 or 2 transaminase elevation, 1 patient defaulted additional ritonavir due to nausea and 1 patient developed diarrhea requiring dose reduction. Viral loads after tuberculosis treatment were available for 11 patients and 10 were undetectable.

Conclusion

Once established on treatment, adjusted doses of LPV/r co-administered with rifampicin-based tuberculosis treatment were tolerated and LPV pre-dose concentrations were adequate.     

The secreted lipoprotein, MPT83, of Mycobacterium tuberculosis is recognized during human tuberculosis and stimulates protective immunity in mice

The long-term control of tuberculosis (TB) will require the development of more effective anti-TB vaccines, as the only licensed vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), has limited protective efficacy against infectious pulmonary TB. Subunit vaccines have an improved safety profile over live, attenuated vaccines, such as BCG, and may be used in immuno-compromised individuals. MPT83 (Rv2873) is a secreted mycobacterial lipoprotein expressed on the surface of Mycobacterium tuberculosis. In this study, we examined whether recombinant MPT83 is recognized during human and murine M. tuberculosis infection. We assessed the immunogenicity and protective efficacy of MPT83 as a protein vaccine, with monophosphyl lipid A (MPLA) in dimethyl-dioctadecyl ammonium bromide (DDA) as adjuvant, or as a DNA vaccine in C57BL/6 mice and mapped the T cell epitopes with peptide scanning. We demonstrated that rMPT83 was recognised by strong proliferative and Interferon (IFN)-γ-secreting T cell responses in peripheral blood mononuclear cells (PBMC) from patients with active TB, but not from healthy, tuberculin skin test-negative control subjects. MPT83 also stimulated strong IFN-γ T cell responses during experimental murine M. tuberculosis infection. Immunization with either rMPT83 in MPLA/DDA or DNA-MPT83 stimulated antigen-specific T cell responses, and we identified MPT83(127-135) (PTNAAFDKL) as the dominant H-2(b)-restricted CD8(+) T cell epitope within MPT83. Further, immunization of C57BL/6 mice with rMPT83/MPLA/DDA or DNA-MPT83 stimulated significant levels of protection in the lungs and spleens against aerosol challenge with M. tuberculosis. Interestingly, immunization with rMPT83 in MPLA/DDA primed for stronger IFN-γ T cell responses to the whole protein following challenge, while DNA-MPT83 primed for stronger CD8(+) T cell responses to MPT83(127-135). Therefore MPT83 is a protective T cell antigen commonly recognized during human M. tuberculosis infection and should be considered for inclusion in future TB subunit vaccines.     

广泛耐药结核分枝杆菌耐药机制及其疾病诊断的研究进展

自20世纪90年代以来,全球结核病疫情回升,结核分枝杆菌耐药是其中的一个重要原因.广泛耐药结核病是指在耐多药结核病(即同时对异烟肼和利福平耐药的结核分枝杆菌引起的结核病)的基础上,还对氟喹诺酮类药物和至少3种二线静脉用抗结核药物(卷曲霉素、卡那霉素、阿米卡星)中的1种耐药的结核分枝杆菌引起的结核病.我国是结核病高流行国...     

Ebola,COVID‐19 and Africa: What we expected and what we got

Democratic Republic of the Congo’s fight with Ebola was just settling when WHO declared COVID‐19 to be a global pandemic on March 12, 2020. This has caused concomitant setbacks in the treatment and control of major health issues like HIV, tuberculosis, measles, and malaria in the country. This, coupled with civil unrest and risk to the safety of the health workers, is a 'perfect storm' waiting to unfold. Military contingents as peacekeepers are having the most difficult time, handling the situation, in the wake of risks involved.