TB drug discovery: addressing issues of persistence and resistance

Tuberculosis remains a leading cause of mortality worldwide into the 21st century. Among the main obstacles to the global control of the disease are emerging multi-drug resistant strains and the recalcitrance of persistent infections to treatment with conventional anti-TB drugs. Here we review recent developments in our understanding of some of the pathways involved in a persistent infection and pathogenesis of Mycobacterium tuberculosis, which reveal new targets for drug development. We describe the high-resolution crystal structures of enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, and of the cyclopropane synthases of mycolic acid biosynthesis. Structure-based drug design is now underway with the potential to lead to the development of new anti-tuberculars effective against persistent and resistant Mycobacterium tuberculosis infections.     

顺德地区肺结核病耐药情况分析

目的分析顺德地区肺结核病耐药情况,为制定下一阶段肺结核防治计划提供科学依据。方法选取2014年1月至2014年11月新增涂阳肺结核患者399例,在此过程中复治患者有71例,对就诊患者中疑似感染人员留取夜间痰、清晨痰和即时痰三份标本进行抗酸杆菌涂片检查和培养,培养阳性标本进行菌种鉴定,对结核分枝杆菌进行异烟肼(H)、链霉素(S)、利福平(R)、乙胺丁醇(E)、左氧氟沙星(LFX)、卡那霉素(KM)、对氨基水杨酸(PAS)7种一、二线药物敏感试验。收集患者初治、复治过程中耐药种类、耐药程度,与性别、年龄、治疗类型间的关系等数据,并进行统计学分析。结果无论在初治患者还是复治患者中R、S、H、E均为患者主要耐受性药物,两组单种耐药性比较数据差异不明显,不具有统计学意义(P0.05)。复治患者较初治患者耐多种药物水平有所增加,两组数据比较,差异明显,具有统计学意义(P0.05)。治疗类型和年龄能够影响患者多种药物耐受性,复治和老年患者中多种药物耐受性增加。结论顺德地区耐药和耐多药肺结核防止和控制工作仍不能忽视,应加强宣传教育和管理,以期降低耐药性。     

肺结核并发各种类型肺外结核患者的分布及其耐药情况分析

目的 探讨肺结核并发各种类型肺外结核患者的分布及其耐药情况。方法 选取2013年1月至2015年12月在首都医科大学附属北京胸科医院住院治疗的347例肺结核并发肺外结核患者(男199例,女148例)为研究对象,回顾性分析患者的年龄、性别、肺外结核并发感染部位等临床特征,同时对痰菌培养阳性的结核分枝杆菌临床分离株进行药物敏感性试验。比较分析并发不同类型肺外结核患者的耐药情况。结果 347例肺结核并发肺外结核患者中,中位年龄33(24~52)岁,其中高发年龄为20~39岁,占41.8%(145/347)。常见并发肺外结核的类型依次为:骨关节结核112例(32.3%,112/347),淋巴结结核96例(27.7%,96/347),结核性脑膜炎82例(23.6%,82/347),结核性腹膜炎23例(6.6%,23/347),结核性心包炎9例(2.6%,9/347),肠结核8例(2.3%,8/347),泌尿系结核和结核性多浆膜炎各5例(各占1.4%,5/347),肾结核2例(0.6%,2/347),脾结核、女性盆腔结核、肝结核、附睾结核和鼻结核各1例(各占0.3%,1/347)。并发骨关节结核、淋巴结结核、结核性脑膜炎、结核性腹膜炎和其他类型肺外结核患者的中位年龄分别为:47(26~60)岁、26(20~42)岁、34(25~50)岁、26(20~46)岁和47(31~66)岁;上述各组中位年龄之间比较,差异有统计学意义(χ ²=36.25,P=0.000)。347例患者中有189例患者进行了痰培养,对其中70例细菌培养阳性的临床分离株进行的药敏试验结果显示,肺结核并发肺外结核的总体耐药率为44.3%(31/70),单耐药率、多耐药率和耐多药率分别为11.4%(8/70)、2.9%(2/70)和30.0%(21/70)。 结论 肺结核并发肺外结核的累及部位广泛,其中最为常见的累及部位为骨关节、淋巴结和脑膜。肺结核并发肺外结核的总体耐药率高。     

MDR1 gene expression and drug resistance of AML cells

We investigated the cellular drug resistance to aclarubicin (Acla), cytosine arabinoside (Ara-C), daunorubicin (Dau), doxorubicin (Dox), etoposide (Etop) and mitoxantrone (Mitox) using the MTT assay at time of disease presentation in 93 cases of acute myeloid leukaemia (AML). In 31 cases we concomitantly investigated MDR1 (multiple drug resistance 1 gene) expression (semi-quantitative competitive RT-PCR) of the leukaemic cells. Drug resistance towards Dau, Dox and Etop was correlated to the MDR1 expression of the AML cells ( P  < 0.05) with high MDR1 expression being associated with high drug resistance towards these drugs. Although the data did not allow firm conclusions to be drawn on the correlation between MDR1 expression and drug resistance towards Ara-C and Mitox, the drug resistance towards Acla clearly was not correlated to, or dependent on, the MDR1 expression level of the AML blast cells. In addition, when examining the cross-activities among the six drugs distinct patterns emerged. Thus, high to very high degrees of cross-activity were found to exist between Dau, Dox, Etop and Mitox, whereas Ara-C had moderate cross-activity with the other drugs except Acla, which showed absent to moderate cross-activity with the other drugs. We conclude that MDR1 gene expression is of significance for cellular drug resistance towards specific (MDR1-related) drugs in AML, whereas it is not of significance regarding drug resistance towards other drugs, which is the case with the anthracycline Acla. We suggest that in the place of other more or less complicated ways to circumvent MDR1-mediated drug resistance, Acla may be used to replace Dau, Dox and other MDR1-related drugs if proven as potent as the drug it is to substitute.     

第二次初治培阳结核患者结核菌耐药性调查

目的掌握1992-2000年我国部分地区初治培阳肺结核患者耐药性变化情况,评价防治对策和措施的有效性。方法采用临床流行病学方法,对全国11个省12家结核病医疗及防治单位进行回顾性调查,分析初治耐药种类及耐药率的动态变化。结果初始耐药率、耐1药率和耐多药率分别为34.4%、16.7%、17.7%,与第一次调查结果(46.0%、20.6%、25.4%)比,均有明显下降。第二次调查前后两个时段初始耐药率和耐1药率也有明显下降,分别为37.4%、22.2%和32.8%、13.6%,而耐多药率尤其是M DR率则为明显上升,分别为15.1%、19.1%和4.6%、5.9%。结论DOTS策略能有效控制敏感菌感染的结核病,而耐多药Tb特别是M DR-Tb比率上升问题必须引起高度重视,其预防和治疗应纳入NTP。     

广东省肇庆地区结核病初始耐药发展趋势

目的　本文对肇庆地区1 996～2 0 0 4年新发初治菌阳肺结核病人1 2 4 2例的初始耐药情况进行分析。结果　发现初始耐药率由2 8.4%下降至1 9.4%。耐多药率有上升趋势,由8.9% ,上升为1 2 .7%。结论　提示初治病人需开展药敏试验,以制定最佳的化疗方案,避免耐多药菌的产生和传播。     

Anti TB drug resistance in Tanga,Tanzania:a cross sectional facility base prevalence among pulmonary TB patients

Objective:To determine the prevalence and risk factors associated with drug resistance tuberculosis(TB) at facility-base level in Tanga,Tanzania.Methods:A total of 79 Mycobacterium tuberculosis(MTB) isolates included in the study were collected from among372(312 new and 60 previously treated) TB suspects self referred to four TB clinics during a prospective study conducted from November 2012 to January 2013.Culture and drug susceptibility test of the isolates was performed at the institute of medical microbiology and epidemiology of infectious diseases,University hospital,Leipzig,Germany.Data on the patient's characteristics were obtained from structured questionnaire administered to the patients who gave informed verbal consent.Unadjusted bivariate logistic regression analysis was performed to assess the risk factors for drug resistant-TB.The significance level was determined at P0.05.Results:The overall proportions of any drug resistance and MDRTB were 12.7%and 6.3%respectively.The prevalence of any drug resistance and MDRTB among new cases were 11.4%and 4.3%respectively,whereas among previously treated cases was 22.2%respectively.Previously treated patients were more likely to develop anti-TB drug resistance.There was no association between anti-TB drug resistances(including MDRTB) with the risk factors analysed.Conclusions:High proportions of anti TB drug resistance among new and previously treated cases observed in this study suggest that,additional efforts still need to be done in identifying individual cases at facility base level for improved TB control programmes and drug resistance survey should continuously be monitored in the country.     

Observations on categorisation of new TB cases: implications for controlling drug resistance.

In the context of rising rates of drug-resistant tuberculosis (TB) in India, this communication presents some field observations during screening of new cases registered with the Revised National Tuberculosis Control Programme (RNTCP) in urban and rural areas of Maharashtra, India. It appears that erroneous categorisation and treatment that contributes to multiple drug resistance results from a lack of patient screening for previous treatment, ambiguity in categorisation and reluctance to disclose a history of anti-tuberculosis treatment. Suggested measures include detailed screening of new cases, computerisation of patient records and an empathetic dialogue between patient and health care provider.     

Microbial resistance to drug therapy: A review

Microbial resistance to the antimicrobials in standard use is becoming more prevalent. A historical perspective frames further discussion. Bacterial resistance is most common, but resistance has been identified in fungi, viruses, and parasites. Resistance is a complex phenomenon that involves the microorganism, the environment, and the patient—separately and interactively. Resistance may be a characteristic of the microbe before exposure to a given drug or may arise as a consequence of therapy. Mechanisms of resistance to antibiotics are discussed. Antibiotic resistance is considered in both hospital and community settings, as ecosystems that are separate yet blending, and the major organisms demonstrating significant resistance problems are presented. A review of existing guidelines, strategies, and plans for addressing resistance and some recommendations conclude this review.     

Expression of multidrug resistance (MDR) proteins and in vitro drug resistance in acute leukemias

The expression of the multidrug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 +/- 2.320) compared to adult AML samples (0.021 +/- 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 +/- 0.529) compared to childhood ALL samples (0.100 +/- 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 +/- 1.760) and ara-C (1.310 +/- 2.390), which was higher compared to childhood AML samples (0.129 +/- 0.214 and 0.210 +/- 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.     

Bacterial drug resistance in meat animals: a review

Prolonged oral or parenteral administration of antibiotics has led to the development of resistant strains of microorganisms. Bacteria acquire drug resistance by mutation, conjugation and transduction. Oral antibiotics by a process of selection pressure facilitate the proliferation of resistant population of bacteria. Drug resistant bacteria are capable of transferring their resistance to drugs to other bacteria by the process of transferable drug resistance (TDR). This can lead to multiple resistance to a vast number of therapeutically useful antibiotics which will, therefore, become ineffective for treatment. TDR can occur between pathogenic organism, between organism of different species, such as E. coli, Salmonella and Shigella; and also between pathogenic and non-pathogenic organisms. Faecal contamination of meat during slaughter may result in the transfer of antibiotic resistant E. coli to the meat. In the human gut this E. coli could transfer resistance to other gut flora, namely E. coli or Salmonella. Antibiotic-resistant coliforms have been isolated from carcases, fresh and cooked meat, raw meat handlers and livestock handlers. Handling of raw market meat by buyers in Nigeria could also lead to contamination of meat with resistant microorganisms. Veterinary drugs are sold and used without much control in Nigeria. This practice may have created a population of resistant bacteria in the meat animals. The presence of antibiotic residues in meat, milk and their products pose potential health hazards for man. Allergic skin conditions, nausea, vomiting, anaphylactic shock and even death have resulted from the ingestion of residues. Cooking and freezing have minimal effect on residues. Resistance to antibiotics have been detected in food poisoning bacteria, namely Salmonella typhimurium, Staphylococcus aureus and Clostridium perfringens. Some epidemiological link has been established between S. typhimurium of calves and food poisoning in man. Judicious use of antibiotics, public education on the health risks of the promiscuous use of drugs in livestock production; and hygienic slaughter at the slaughter houses, will help to reduce bacterial drug resistance in man and animals.     

武汉市涂阳肺结核患者耐药率及耐药谱分析

目的 分析不同登记分类涂阳肺结核患者的耐药［特别是耐多药结核病（MDR-TB）］特点,了解耐药的危险因素,为制订耐药结核病（特别是MDR-TB）控制策略提供参考。 方法 对痰涂片阳性的新患者、复治（复发、返回和其他）和治疗失败（初治失败、复治失败和治疗3个月末痰涂片阳性的新患者）3类患者收集2份痰标本进行培养,用比例法进行6种抗结核药物（INH、RFP、EMB、S、Km和Ofx）敏感性试验。2006年12月至2012年3月进行痰培养的涂阳肺结核患者2110例,培养阳性1947例（92.3%）;经药物敏感性试验和菌型鉴定,Mtb感染1800例（92.4%）。 结果 1800例Mtb感染者中总耐药794例（44.1%,794/1800）,其中单耐药结核病（SDR-TB） 244例（13.6%, 244/1800）、多耐药结核病（PDR-TB）155例（8.6%,155/1800）、MDR-TB 395例（21.9%,395/1800）;MDR-TB检出率明显高于SDR-TB和PDR-TB（χ²=43.4,χ²=123.6,P值均＜0.01）。新患者、复治及治疗失败3类患者SDR-TB检出率分别为11.9%（57/480）、14.4%（159/1107）及13.1%（28/213）,差异无统计学意义（χ²=0.33,P=0.85）;3类患者PDR-TB检出率分别为5.4%（26/480）、9.8%（109/1107）及9.4%（20/213）,复治与治疗失败PDR-TB检出率差异无统计学意义（χ²=0.04,P=0.84）,但高于新患者（χ²=8.53,P=0.01）;3类患者MDR-TB检出率分别为14.4%（69/480）、22.5%（249/1107）及36.2%（77/213）,经χ²检验,治疗失败患者MDR-TB检出率高于复治和新患者（χ²=17.92,χ²=42.07,P值均＜0.01）。 结论 武汉市涂阳肺结核患者耐药检出率高;耐药成因复杂,耐药谱呈多样性和复杂性,对结核病控制构成威胁。     

结核病耐药性检测技术进展及存在的问题和对策

结核病耐药性检测技术研究取得了显著进展,但仍不能满足临床诊治及防控的需求。一些新的细菌学和分子生物学药敏检测技术尽管比传统的耐药性检测方法更加快速和灵敏,但是各种新技术在临床应用中也存在着不少问题。就这些新技术的优缺点及可能解决的措施和进展情况提出了一些看法,以便促进这些新技术的推广和应用。     

耐多药肺结核病人痰菌耐药不均一性研究报告

目的　探讨耐多药肺结核病人痰菌是否存在耐药不均一性。方法　取 1例耐多药肺结核病人痰分离培养物制成的菌悬液 ,用 8 0 μm孔径醋酸纤维膜过滤得到单条结核菌分散存在的菌悬液 ,稀释后接种培养 ,挑取2 4个单菌落 ,再分别传代培养获得 2 4个结核菌的纯种。再采取中国标准绝对浓度法对 2 4个纯种菌株进行药物(INH、RFP、SM、EMB)敏感性试验 ,以敏感株H37Rv为对照。结果　从耐多药肺结核病人的分离株得到的 2 4个纯种结核菌中 ,对INH敏感 1个 ( 4 2 % )、低耐 2 2个 ( 91 6% )、高耐 1个 ( 4 2 % ) ;对RFP敏感 2个 ( 8 3 % )、低耐 2个( 8 3 % )、高耐 2 0个 ( 83 4% ) ;对SM敏感 1个 ( 4 2 % )、低耐 0个 ( 0 % )、高耐 2 3个 ( 95 8% ) ;对EMB全部敏感。结论　耐多药肺结核病人的痰菌对多种抗结核药物均存在耐药不均一性 ,敏感菌与耐药菌、低耐菌与高耐菌混合存在 ,敏感菌为极少数 ,耐药菌占绝大多数     

Alcohol and drug use disorders, HIV status and drug resistance in a sample of Russian TB patients.

SETTING: Alcohol use, tuberculosis (TB) drug resistance and human immunodeficiency virus (HIV) risk behavior are of increasing concern in Russian TB patients. DESIGN: A prevalence study of alcohol use and HIV risk behavior was conducted in a sample of 200 adult men and women admitted to TB hospitals in St Petersburg and Ivanovo, Russia. RESULTS: Of the subjects, 72% were men. The mean age was 41. Active TB was diagnosed using a combination of chest X-ray, sputum smears and sputum cultures. Sixty-two per cent met DSM-IV criteria for current alcohol abuse or dependence. Drug use was uncommon, with only two patients reporting recent intravenous heroin use. There was one case of HIV infection. The mean total risk assessment battery score was 3.4. Depression was present in 60% of the sample, with 17% severely depressed. Alcohol abuse/dependence was associated with an eight-fold increase in drug resistance (OR 8.58; 95% CI 2.09-35.32). Patients with relapsing or chronic TB were more likely to meet the criteria for alcohol abuse/dependence (OR 2.56; 95% CI 1.0-6.54). CONCLUSION: Alcohol use disorders are common in patients being treated for active TB, and are associated with significant morbidity. Additional surveys are needed to examine the relationship between alcohol use disorders and anti-tuberculosis drug resistance.     

耐多药/广泛耐药肺结核并发2型糖尿病患者耐药特征分析

目的 了解并分析耐多药/广泛耐药肺结核并发2型糖尿病患者的耐药特征。方法 搜集黑龙江省传染病防治院耐药病区 2018年6月至2020 年6月收治的经结核分枝杆菌培养及药物敏感性试验(简称“药敏试验”)确诊的耐多药/广泛耐药肺结核患者233例,其中并发2型糖尿病者84例(简称“并发糖尿病组”),未并发糖尿病者149例(简称“未并发糖尿病组”)。对两组患者的肺结核初复治情况、所耐药品数量、耐药谱进行分析,采用SPSS 18.0软件进行统计学分析,组间计数资料“构成比或率”的比较采用 χ²检验,以P<0.05为差异有统计学意义。 结果 并发糖尿病组初治肺结核26例,占比为30.95%(26/84);未并发糖尿病组初治肺结核16例,占比为10.74%(16/149),两组比较差异有统计学意义(χ²=14.854,P<0.01)。在所耐药品数量上,并发糖尿病组与未并发糖尿病组比较,耐异烟肼与利福平的构成比分别为10.71%(9/84)、7.39%(11/149),耐3种药品的构成比分别为28.57%(24/84)、24.16%(36/149),耐4种药品的构成比分别为19.05%(16/84)、24.83%(37/149),耐5种药品的构成比分别为17.86%(15/84)、19.46%(29/149),耐6种及以上药品的构成比分别为23.81%(20/84)、24.16%(36/149);两组间差异均无统计学意义(χ²值分别为0.759、0.546、1.023、2.063、0.882,P值分别为0.383、0.460、0.312、0.151、0.348)。在耐药谱方面,并发糖尿病组与未并发糖尿病组比较,异烟肼与利福平耐药的比率分别为10.71%(9/84)、7.40%(11/149),氟喹诺酮类药品耐药的比率分别为29.76%(25/84)、29.53%(44/149),抗结核药品二线注射剂耐药的比率分别为4.76%(4/84)、6.71%(10/149),异烟肼与利福平+氟喹诺酮类药品+抗结核药品二线注射剂同时耐药的比率分别为8.33%(7/84)、13.42%(20/149),两组间差异均无统计学意义(χ²值分别为0.760、0.009、0.361、1.358,P值分别为0.383、0.926、0.548、0.244)。结论 耐多药/广泛耐药肺结核并发2型糖尿病患者以初治肺结核多见,是否并发糖尿病对耐多药/广泛耐药肺结核所耐药品数量、耐药谱均没有影响。对糖尿病等易感人群要加强防护,避免因感染耐药结核分枝杆菌而发生原发性耐药结核病。