Drug resistance mechanisms and novel drug targets for tuberculosis therapy

Drug-resistant tuberculosis （TB） poses a significant challenge to the successful treatment and control of TB worldwide. Resistance to anti-TB drugs has existed since the beginning of the chemotherapy era. New insights into the resistant mechanisms of anti-TB drugs have been provided. Better understanding of drug resistance mechanisms helps in the development of new tools for the rapid diagnosis of drug- resistant TB. There is also a pressing need in the development of new drugs with novel targets to improve the current treatment of TB and to prevent the emergence of drug resistance in Mycobacterium tuber- culosis. This review summarizes the anti-TB drug resistance mechanisms, furnishes some possible novel drug targets in the development of new agents for TB therapy and discusses the usefulness using known targets to develop new anti-TB drugs. Whole genome sequencing is currently an advanced technology to uncover drug resistance mechanisms in M. tuberculosis. However, further research is required to unravel the significance of some newly discovered gene mutations in their contribution to drug resistance.

Application of artificial intelligence in digital chest radiography reading for pulmonary tuberculosis screening

Currently, the diagnosis of tuberculosis (TB) is mainly based on the comprehensive consideration of the patient’s symptoms and signs, laboratory examinations and chest radiography (CXR). CXR plays a pivotal role to support the early diagnosis of TB, especially when used for TB screening and differential diagnosis. However, high cost of CXR hardware and shortage of certified radiologists poses a major challenge for CXR application in TB screening in resource limited settings. The latest development of artificial intelligence (AI) combined with the accumulation of a large number of medical images provides new opportunities for the establishment of computer-aided detection (CAD) systems in the medical applications, especially in the era of deep learning (DL) technology. Several CAD solutions are now commercially available and there is growing evidence demonstrate their value in imaging diagnosis. Recently, WHO published a rapid communication which stated that CAD may be used as an alternative to human reader interpretation of plain digital CXRs for screening and triage of TB.

Occurrence of Multi-Drug Resistant <i>Listeria species</i>in Faecal Samples of Poultry Chickens in Rural Farms in Lagos State, Nigeria

Background: Listeriosis is a common zoonotic disease caused by a foodborne pathogen, Listeria monocytogenes. Poultry meat and products have been established as vehicles of transmission of pathogenic Listeria strains to humans. This study evaluates the occurrence of Listeria species in faeces of poultry chicken in Lagos. Methods: One hundred and fourteen pooled fresh faecal samples from cage-reared broiler chickens were collected from 12 farms in three rural areas in Lagos State from May to August 2019. All samples were analysed for Listeria species detection according to ISO11290-1 standard and confirmed using PCR assay. Susceptibility testing was performed using the Kirby-Bauer disc diffusion technique. Results: Twenty-eight (24.6%) Listeria species were detected from 114 faecal samples. The isolated Listeria species were L. monocytogenes 8 (7.0%), L. ivanovii 9 (7.9%), L. grayi 7 (6.1%) and L. innocua 4 (3.5%). There was no significant difference in the frequency of occurrence of Listeria species across the different locations (X2 = 4.98, p = 0.08). The listeria species were susceptible to Augmentin (96.4%), vancomycin (85.7%) and co-trimoxazole (82.1%), but resistant to ceftazidime (100%), tetracycline (75.0%) and ciprofloxacin (71.4%). Conclusion: This study reveals high occurrence of multi-drug resistant Listeria species in faecal samples of poultry chickens in Lagos state which may be an important vector in the contamination of the environment and transmission of antibiotic resistant Listeria species to consumers.

研究如何能帮助结核病控制

自从科赫发现结核菌后,结核病(TB)在生物医学研究史上发挥了举足轻重的作用。19世纪和20世纪的研究产出了非常有价值的控制结核病的诊断、治疗和预防工具。随着上世纪七八十年代短程化疗的发展,结核病的研究实际上逐渐消失了。即使存在着一批工具,结核病控制还是失败了,这种疾病仍然是一个主要的致死原因。控制结核病科学研究成果的失败是控制方法本身的缺陷以及在结核病高负担人群中应用不足造成的。一个不断变化的流行状态,与人类免疫缺陷病毒相关的结核病疫情不断上升以及耐多药结核病的出现,给全球结核病带来进一步威胁。为了实现21世纪结核病控制的全球目标,需要一个强有力的研究计划。需要基础研究来帮助我们更好地理解结核病病原学和免疫学,并确定诊断、治疗和接种疫苗的目标。因为所有现存的方法都有很明显的缺陷,关于发现、治疗和预防结核病的更好的生物医学工具的研究也是一个主要的优先领域。此外还急需关于理解如何在人群中将现存方法和新方法同时应用于结核病控制方面的研究。2007年全球用于结核病研究的资金有4.83亿美元,数额虽然缓慢增长但是仍远远低于所需。为实现遏制结核病全球计划的远大目标和千年发展目标,需要在研究方面投入巨额经费。

活动性肺结核患者接触者中的结核病发病率

背景:在巴西,对于潜伏结核感染(LTBI)的治疗只应用于那些接触痰菌涂阳患者、年龄小于15岁、结核菌素皮试(TST)结果≥10mm、没有接种过卡介苗(BCG)的人群以及那些无论是否接种过BCG但TST结果≥15mm的接触者。目的:评估2年发病率和不符合巴西LTBI治疗标准的结核病接触者中的一些预测指标。设计:人群回顾性研究。12～15岁接触者以及那些>15岁但不符合巴西LTBI治疗标准者进入研究范围。结果:结核病发病率为3.2%(22/667),估计结核病发病率为1649/10万;349例TST结果≥5mm接触者(5.4%)发病的危险性高于318例TST结果<5mm接触者(0.9%;RR6.04,95%CI1.7～20.6)结论:不符合巴西LTBI治疗标准的接触者中,如此高的发病率强烈建议这些标准应重新考虑。另外,即使接种过BCG的接触者,TST硬结≥5mm是唯一的可以预测在2年内发病的变量。

Design,synthesis,and biological evaluation of 1,2,4-triazole derivatives as potent antitubercular agents

Inhibition of mycobacterial membrane protein large 3(MmpL3)thereby affecting the mycolic acid biosynthetic pathway has been proven to be an effective strategy for developing antitubercular drugs.Based on the X-ray crystal structure of MmpL3 inhibitor complexes,a series of novel 1,2,4-triazole derivatives were designed,synthesized and evaluated antitubercular activity against Mtb strain H37Rv.Comprehensive structure–activity relationship exploration resulted in the identification of compounds 21 and 28,which possess potent antitubercular activity against Mtb strain H37Rv[minimum inhibitory concentration(MIC)=0.03–0.13μg/mL]and the clinical isolates of multidrug resistance(MDR)and extensive drug resistance(XDR)tuberculosis(MIC=0.06–1.0μg/mL).Moreover,compounds 21 and 28 showed neglectable cytotoxicity(IC_(50)≥32μg/mL)to the mammalian Vero cells and favorable physicochemical and pharmacokinetic properties according to the in silico absorption,distribution,metabolism and excretion(ADME)prediction.Finally,the potential target of representative 1,2,4-triazole 28 was identified to be MmpL3 using a microscale thermophoresis(MST)assay.

Innate immunity in tuberculosis: host defense vs pathogen evasion

The major innate immune cell types involved in tuberculosis(TB)infection are macrophages,dendritic cells(DCs),neutrophils and natural killer(NK)cells.These immune cells recognize the TB-causing pathogen Mycobacterium tuberculosis(Mtb)through various pattern recognition receptors(PRRs),including but not limited to Toll-like receptors(TLRs),Nod-like receptors(NLRs)and C-type lectin receptors(CLRs).Upon infection by Mtb,the host orchestrates multiple signaling cascades via the PRRs to launch a variety of innate immune defense functions such as phagocytosis,autophagy,apoptosis and inflammasome activation.In contrast,Mtb utilizes numerous exquisite strategies to evade or circumvent host innate immunity.Here we discuss recent research on major host innate immune cells,PRR signaling,and the cellular functions involved in Mtb infection,with a specific focus on the host’s innate immune defense and Mtb immune evasion.A better understanding of the molecular mechanisms underlying host–pathogen interactions could provide a rational basis for the development of effective anti-TB therapeutics.

Molecular epidemiology of Mycobacterium tuberculosis in Gansu province of China

Background Mycobacterial interspersed repetitive units-variable number tandem repeat （MIRU-VNTR） and Beijing family typing based on detecting the deletion of RD105 sequence are two common genotyping methods used to study the molecular epidemiologic characteristics of Mycobacterium （M.） tuberculosis. We collected 218 strains of M. tuberculosis between 2004 and 2006 in the Linxia Hui Autonomous Prefecture of Gansu province in Northwest China. Methods MIRU-VNTR analysis and Beijing family typing based on detecting the deletion of RD105 sequence were used to type the 218 strains, and their typing power was evaluated to look for practical and efficient genotyping methods suitable for the region. Results The MIRU typing yielded 115 distinct genotypes, including 98 unique isolates and 17 different clusters containing 120 isolates （55.05%）; the cluster rate was 47.25%. By detecting the deletion of RD105 sequence, 188 of 218 （86.23%） isolates belonged to Beijing family. Combination of Beijing family typing and MIRU typing yielded 118 distinct patterns, including 101 unique isolates and 17 clusters containing 117 isolates （54.13%）. The largest cluster contained 58 strains with MIRU genotype of 223325173533 which contained 50 strains belonging to Beijing family and 8 strains belonging to non-Beijing family. Conclusions The Beijing family strains occupied a large proportion and the Beijing family MIRU genotype 223325173533 is a dominant strain in Linxia of Gansu. Combining detecting the deletion of RD105 and MIRU typing together provides a simple, fast, and effective method which is low in cost and might be practical and suitable for M. tuberculosis aenotvDina in China.

Mycobacterium tuberculosis latency-associated antigen Rv1733c SLP improves the accuracy of differential diagnosis of active tuberculosis and latent tuberculosis infection

Background: Differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) has been a challenge for clinicians in high TB burden countries. The purpose of this study was to improve the accuracy of differential diagnosis of ATB and LTBI by using fluorescent immunospot (FluoroSpot) assay to detect specific Th1 cell immune responses. The novelmycobacterium tuberculosis (MTB) latency-associated antigens Rv1733c and synthetic long peptides derived from Rv1733c (Rv1733c SLP) were used based on virulence factors early secreting antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10).Methods: Fifty-seven ATB cases, including 20 pathogen-confirmed ATB and 37 clinically diagnosed ATB, and 36 LTBI cases, were enrolled between January and December 2017. FluoroSpot assay was used to detect the interferon γ (IFN-γ) and interleukin 2 (IL-2) secreted by the specific T cells after being stimulated with MTB virulence factors ESAT-6 and CFP-10, MTB latency-associated antigens Rv1733c and Rv1733c SLP. The receiver operating characteristic (ROC) curve was used to define the best cutoff value of latency-associated antigens in the use of differentiating ATB and LTBI. The sensitivity, specificity, predictive value, and likelihood ratio of ESAT-6 and CFP-10-FluoroSpot combined with latency-associated antigen in the differential diagnosis of ATB and LTBI were also calculated.Results: Following the stimulation with Rv1733c and Rv1733c SLP, the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP had the largest area under the ROC curve, which was 0.766. With a cutoff value of 1 (spot-forming cells [SFCs]/2.5 × 105 peripheral blood mononuclear cells) for frequency, the sensitivity and specificity of distinguishing ATB from LTBI were 72.2% and 73.7%, respectively. ESAT-6 and CFP-10-FluoroSpot detected the frequency and proportion of single IFN-γ-secreting T cells;the sensitivity and specificity of distinguishing ATB from LTBI were 82.5% and 66.7%, respectively. Combined with the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP on the basis of ESAT-6 and CFP-10-FluoroSpot, the sensitivity and specificity increased to 84.2% and 83.3%, respectively.Conclusion: Rv1733c SLP, combined with ESAT-6 and CFP-10, might be used as a candidate antigen for T cell-based tuberculosis diagnostic tests to differentiate ATB from LTBI.

Distinct lung microbial community states in patients with pulmonary tuberculosis

An improved understanding of the lung microbiome may lead to better strategies to diagnose, treat, and prevent pulmonary tuberculosis (PTB). However, the characteristics of the lung microbiomes of patients with TB remain largely undefined. In this study, 163 bronchoalveolar lavage (BAL) samples were collected from 163 sputum-negative suspected PTB patients. Furthermore, 12 paired BAL samples were obtained from 12 Mycobacterium tuberculosis-positive (MTB+) patients before and after negative conversion following a two-month anti-TB treatment. The V3–V4 region of the 16S ribosomal RNA (rRNA) gene was used to characterize the microbial composition of the lungs. The results showed that the prevalence of MTB in the BAL samples was 42.9% (70/163) among the sputum-negative patients. The α-diversity of lung microbiota was significantly less diverse in MTB+ patients compared with Mycobacterium tuberculosis-negative (MTB–) patients. There was a significant difference in β-diversity between MTB+ and MTB– patients. MTB+ patients were enriched with Anoxybacillus, while MTB– patients were enriched with Prevotella, Alloprevotella, Veillonella, and Gemella. There was no significant difference between the Anoxybacillus detection rates of MTB+ and MTB– patients. The paired comparison between the BAL samples from MTB+ patients and their negative conversion showed that BAL negative-conversion microbiota had a higher α-diversity. In conclusion, distinct features of airway microbiota could be identified between samples from patients with and without MTB. Our results imply links between lung microbiota and different clinical groups of active PTB.

Cytomegalovirus antigen-specific T cell immune responses in patients with autoimmune diseases under different cytomegalovirus infection status

To the Editor:Patients with autoimmune diseases have increased risk of active cytomegalovirus(CMV)infection due to immune dysfunction or the application of glucocorticoid and immunosuppressants.[1]Cui et al[2]found that compared with human immunodeficiency virus(HIV)/acquired immune deficiency syndrome(27%),posttransplant patients(14.8%),and hematological malignancies(5.1%),patients with autoimmune disease had the highest incidence of CMV antigenemia(35.1%).Monitoring CMV antigen-specific T cell immunity is helpful for understanding the protective immunity against CMV infection and identifing the risk of CMV-related complications.Currently,the CMV antigen-specific T cell immune responses in patients with autoimmune diseases remains unknown.

A highly efficient in vivo plasmid editing tool based on CRISPR-Cas12a and phage λ Red recombineering

Plasmids are useful tools for studying genetic information in living cells,as well as heterologous expression of genes and pathways in cells(Lauritsen et al.,2018).Various methods have been developed for plasmid manipulation both in vivo and in vitro(Aslanidis and de Jong,1990;Li and Elledge,2007;Xia et al.,2018).However,large plasmids,such as P1-based artificial chromosomes(PACs),bacterial artificial chromosomes(BACs),and fosmids,are difficult to manipulate.