A novel nucleotide-containing antigen for human blood γδ T lymphocytes

The stimulation of human γδ T cells by mycobacteria occurs through recognition of four distinct nonpeptide phosphorylated antigens termed TUBag1–4. Among these latter, TUBag4 has already been biochemically characterized as a γ-X derivative of 5′-deoxythymidine triphosphate (Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M. and Fournié, J.-J., Science 1994. 264: 267). However, despite chemical synthesis of weakly stimulatory nucleotide-containing analogs, these mycobacterial compounds remained the sole nucleotide-containing antigens actually isolated from natural sources. Here, we present the complete isolation of the TUBag3 antigen from Mycobacterium fortuitum and demonstrate that this nonpeptide molecule contains a 5′-UTP nucleotide moiety. On selected Vγ9/Vδ2 clones, T cell responses can be triggered with nanomolar concentrations of TUBag3. Like crude mycobacterial extracts, this purified nucleotide conjugate elicits a strong polyclonal response of γδ PBL from healthy donors. Furthermore, we present evidence that this compound is distinct from the recently synthesized γ-isopentenyl 5′-UTP, a nucleotide conjugate of isopentenyl pyrophosphate that was found to be stimulatory for human γδ T cells (Tanaka, Y., Morita, C. T., Tanaka, Y., Nieves, E., Brenner, M. B. and Bloom, B. R., Nature 1995. 375: 155). Since it appears that both mycobacterial nucleotide antigens are molecules structurally related to peculiar precursors of nucleic acid synthesis, we propose that TUBag-reactive T cells might be specifically devoted to surveillance of proliferating cells.     

Interferon-gamma receptor 1 promoter polymorphisms: population distribution and functional implications

Different polymorphisms have been described in the minimal promoter region (MPR) of the interferon-gamma receptor 1 (IFNGR1), a molecule that plays a critical role in mycobacterial control. We sequenced the IFNGR1 MPR from African American, Caucasian and Korean controls, and from mycobacteria-infected patients. Six different single nucleotide polymorphisms (SNPs) were detected in the IFNGR1 MPR. The three ethnic groups showed different SNP distribution patterns, but no significant differences were detected between mycobacterial cases and controls. Two polymorphisms were found in all populations (G-611A, T-56C). We cloned the four allelic variants (var) of haplotype G-611A/T-56C into a luciferase reporter vector and determined their promoter activity. Polymorphisms at position -611 had a stronger effect on the promoter activity than those at position -56, and constructs carrying G-611 produced a stronger promoter activity than -611A constructs. The IFNGR1 MPR is a polymorphic region with at least two SNPs influencing its activity, but these are not associated with increased mycobacterial susceptibility.     

老年支气管扩张合并非结核分枝杆菌感染的临床特征及其危险因素

目的分析老年支气管扩张合并非结核分枝杆菌(non-tuberculous mycobacterial,NTM)的临床特征及其危险因素。方法选择2015年1月-2019年12月无锡市第五人民医院呼吸与危重症科收治的老年支气管扩张患者为研究对象,其中120例合并NTM感染患者为合并感染组,120例为单纯支气管扩张组。分析合并NTM感染患者临床症状及影像学特点,采集痰液标本进行分枝杆菌鉴定及药敏试验,单因素及多因素Logistic回归分析支气管扩张合并NTM的危险因素。结果 120例合并NTM感染患者主要症状为咳嗽咯痰、咯血;影像学检查除典型的支气管扩张影外,还伴随有斑点、斑块、条索影,同时还有部分结节影、薄壁空洞表现,并且以1~2种征象为主,多种表现混合存在;累及3个以上肺野的患者占78.33%;痰抗酸杆菌涂片阳性率为86.67%(104/120)、痰NTM-DNA检测阳性率为90.00%(108/120);Ⅲ组鸟-胞内分枝杆菌复合菌群54例占45.00%,Ⅳ组龟-脓肿分枝杆菌复合菌群58例占48.33%,其他复合菌8例占6.67%;120株NTM对一、二线抗结核药物耐药率均较高,且全耐药者达到了38.33%(46/120);吸烟史>20年、支扩累及肺叶数≥5叶、薄壁空洞、CD₄⁺<550个/ml、低蛋白血症是老年支气管扩张合并NTM感染的独立危险因素(P<0.05)。结论老年支气管扩张合并NTM感染患者临床症状及影像学表现与肺结核相似,但对抗结核药物高度耐药,临床上可根据痰抗酸杆菌涂片及NTM-DNA检测对NTM感染进行判别,同时合并NTM感染受多种因素影响。     

Specific and nonspecific lymphadenitis in childhood: etiology, diagnosis, and therapy

Over a period of 4 years, 39 children with lymphadenitis were treated surgically; in 31 cases cervical lymph nodes were the main location. In 9 cases the lymphadenitis was caused by mycobacterial infection.Staphylococcus aureus was the most frequent causative organism of unspecific lymphadenitis (11 cases). The therapy of choice appears to be surgical treatment and medical care after operation. Especially in mycobacterial lymphadenitis, complete surgical excision of the lymph node is decisive for definitive healing. There was only 1 case of therapy-resistant, relapsing cervical lymphadenitis that needed a second operation. Causative organisms in this case wereMycobacterium avium andMycobacterium intracellulare. All other patients showed an uneventful postoperative clinical course. We believe that a consequent diagnostic process and cooperation between the pediatric surgeon and pediatrician are necessary for effective therapy.     

Performance of lipid fingerprint-based MALDI-ToF for the diagnosis of mycobacterial infections

ObjectivesBacterial diagnosis of mycobacteria is often challenging because of the variability of the sensitivity and specificity of the assay used, and it can be expensive to perform accurately. Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become the workhorse of clinical laboratories, the current MALDI methodology (which is based on cytosolic protein profiling) for mycobacteria is still challenging due to the number of steps involved (up to seven) and potential biosafety concerns. Knowing that mycobacteria produce surface-exposed species-specific lipids, we here hypothesized that the detection of those molecules could offer a rapid, reproducible and robust method for mycobacterial identification.MethodsWe evaluated the performance of an alternative methodology based on characterized species-specific lipid profiling of intact bacteria, without any sample preparation, by MALDI MS; it uses MALDI-time-of-flight (ToF) MS combined with a specific matrix (super-2,5-dihydroxybenzoic acid solubilized in an apolar solvent system) to analyse lipids of intact heat-inactivated mycobacteria. Cultured mycobacteria are heat-inactivated and loaded directly onto the MALDI target followed by addition of the matrix. Acquisition of the data is done in both positive and negative ion modes. Blinded studies were performed using 273 mycobacterial strains comprising both the Mycobacterium tuberculosis (Mtb) complex and non-tuberculous mycobacteria (NTMs) subcultured in Middlebrook 7H9 media supplemented with 10% OADC (oleic acid/dextrose/catalase) growth supplement and incubated for up to 2 weeks at 37°C.ResultsThe method we have developed is fast (<10 mins) and highly sensitive (<1000 bacteria required); 96.7% of the Mtb complex strains (204/211) were correctly assigned as MTB complex and 91.7% (22/24) NTM species were correctly assigned based only on intact bacteria species-specific lipid profiling by MALDI-ToF MS.ConclusionsIntact bacterial lipid profiling provides a biosafe and unique route for rapid and accurate mycobacterial identification.     

安徽省420株结核分枝杆菌对一线和二线抗结核药物药敏结果分析

目的研究并分析安徽地区420例结核分枝杆菌对一线、二线抗结核药物菌耐药情况，为结核病临床治疗和结核病控制提供科学依据。方法采用绝对浓度法对420例结核分枝杆菌临床分离株进行一线、二线抗结核药物敏感试验。结果对一线抗结核药物总耐药率和耐多药率分别是29．05％和14．52％；其中初治耐药率和耐多药率分别是15．93％、6．79％，复治耐药率和耐多药率分别是52．86％、30．0％；RIF、INH、SM、EMB耐药率分别是22．14％、20．0％、17．38％和0．95％。抗二线结核药物总耐药率为22．62％，其中左氧氟沙星和丙硫乙烟胺的耐药率较高，分别为13．33％（56／420）和12．86％（54／420），年龄段41～60岁耐药率均显著高于其他年龄段（P〈0．05）；男性和女性病例组间均无统计学显著性差异（P〉0．05）。结论安徽地区结核菌耐药情况仍然十分严重，应加强抗结核药物的耐药性监测，同时根据药敏试验结果选择科学有效的化疗方案。     

Genitourinary non-tuberculous mycobacterial (GU-NTM) infections: A single institution experience in South India

IntroductionNon-tuberculous mycobacterial (NTM) infections are rarely reported, and more so with genitourinary infections. This retrospective study was designed to understand the proportion and behaviour of genitourinary non-tuberculous mycobacterial (GU-NTM) infections compared with genitourinary mycobacterial tuberculosis (GU-MTB) treated at a tertiary care hospital in South India.Materials and methodsThe hospital records of every bacteriologically proved GU-MTB and GU-NTM infections treated at this centre from 2010 to 2016 were retrospectively reviewed.ResultsThere were ten patients of GU-NTM and 15 patients of GU-MTB. There was no significant difference in presentation other than lesser frequency of irritative lower urinary tract symptoms (LUTS) among patients with GU-MTB. Urine smear for AFB was positive in 60% and 47% of GU-NTM and GU-MTB patients. 40% of GU-NTM patients had history of urinary tract instrumentation. Mycobacterium abscessus was grown in four patients and one had Mycobacterium fortuitum/chelonae complex; all the rest were rapid growers. No patient had multi-drug resistant tuberculosis. Imaging studies of GU-NTM patients were indistinguishable from GU-MTB with renal, ureteral and bladder involvements, and stone formation. The drug sensitivities varied among the NTM patients but all showed sensitivity to clarithromycin uniformly. Need for varieties of surgeries in the early and late phases were also comparable.ConclusionsGU-MTB and GU-NTM infections are indistinguishable from their clinical presentation and imaging studies. All cases of suspected genitourinary mycobacterial infections must be subjected to nucleic acid testing. Treatments based on clinical and radiological features without culture studies may misdiagnose GU-NTM infections as MDR GU-MTB, thereby delaying the appropriate treatment.     

Use of MALDI-TOF MS (Bruker Daltonics) for identification of Mycobacterium species isolated directly from liquid medium

ObjectivesThe aim of this study was to describe the evaluation of the use of MALDI-TOF MS for the identification of non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis directly from liquid MGIT cultures from January 2017 to December 2017.Material/methodsA total of 155 isolates (mainly respiratory) were analyzed by MALDI-TOF MS (Bruker Daltonics) directly from MGIT liquid medium with a previous extraction procedure.ResultsMALDI-TOF MS generated acceptable scores for 152 isolates (98.06%). Fifty isolates were identified as M. tuberculosis complex and the remaining 105 as NTM (M. abscessus subsp. abscessus, M. avium, M. celatum, M chelonae, M. chimaera, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. lentiflavum, M. mageritense, M. mucogenicum and M. xenopi).ConclusionsThese results indicate that MALDI-TOF MS can be useful to identify mycobacteria directly from MGIT cultures and is an accurate, rapid and cost-effective system to be used as a routine method.