2014年大连市结核病耐药监测结果分析

摘要：目的 全面了解大连市耐药结核病流行现状和特点，为有效控制耐药结核病提供依据。方法 收集2014年度初治、复治涂阳病例，对成功分离的448例结核分枝杆菌菌株，采用比例法对利福平、异烟肼、链霉素、乙胺丁醇、左氧氟沙星、阿米卡星、对氨基水杨酸、丙硫异烟胺、卷曲霉素9种药物进行药物敏感试验，采用SPSS 12.0进行统计学分析，对耐药率的比较采用卡方检验，P<0.05为差异有统计学意义。结果 分离的448株菌株总体耐药率为26.8%，初治耐药率22.2%，获得性耐药率37.2%，耐多药率为6.9%，初始耐多药率5.5%，获得性耐多药率10.2%。结论 大连市结核病耐药发生率较高，但呈下降趋势。     

广西地区艾滋病患者临床标本分枝杆菌培养及耐药性分析

为探讨艾滋病患者临床标本分枝杆菌培养阳性率、菌种类型及耐药状况,本研究对2010年1月-2019年12月在广西某医院就诊的艾滋病患者的临床标本进行分枝杆菌培养,分离鉴定后用8种以上抗结核药物进行药敏试验。结果显示,艾滋病患者临床标本分枝杆菌培养总阳性率为15.68%(2 163/13 795),脓液、分泌物、组织标本、胸腹水等标本阳性率高。经初步菌种鉴定,结核分枝杆菌复合群占77.95%(1 442/1 850),非结核分枝杆菌占22.05%(408/1 850),后者10年中占比15.71%～26.07%,年间差异无统计学意义(χ2＝10.442,P＞0.05)。结核分枝杆菌的总耐药率为23.30%(336/1 442),年间差异有统计学意义(χ2＝18.901,P＝0.026),其对异烟肼的耐药率为12.14%(175/1 442)、对利福平为10.54%(152/1 442)、对链霉素为9.29%(134/1 442)、对氧氟沙星为5.62%(81/1 442)、对乙胺丁醇为3.05%(44/1 442)、对对氨基水杨酸为1.80%(26/1 442)、对卡那霉素为1.32%(19/1 442),耐多药率、广泛耐药率分别为5.48%(79/1 442)、0.28%(4/1 442)。研究表明,艾滋病患者临床标本分枝杆菌培养阳性率高,其中非结核分枝杆菌占 15.71%～26.07%,10年来基本稳定。结核分枝杆菌对异烟肼耐药率最高,利福平次之。因此,应该对艾滋病患者临床标本进行分枝杆菌培养、鉴定和耐药性检测,从而为临床诊治提供可靠依据。     

201例结核病患者分离株的药敏结果分析

本文旨在分析2008年12月～2009年5月就诊于上海市肺科医院的201例结核病患者结核分枝杆菌的药敏试验结果。结核分枝杆菌培养采用罗氏培养系统,药敏试验采用绝对浓度法。201例患者中,耐药率由高到低依次为异烟肼(INH,52.24%)、链霉素(SM,45.77%)、利福平(RFP,30.35%)、乙胺丁醇(EMB,28.86%)、阿米卡星(AK,28.18%)、利福喷汀(RIB,26.40%)、力克肺疾(Pa,25.56%)、对氨基水杨酸(PAS,23.60%)、丙硫异烟胺(Pt,23.03%)、卷曲霉素(CPM,20.56%)。其中,初治患者的耐药率为3.08%～26.15%,复治患者为27.50%～64.71%,耐多药患者为64.44%～100%。总体来看,与低浓度相比,高浓度SM、INH、EMB、RIB、Pt可明显降低结核分枝杆菌的耐药率(P0.05)。除INH、SM、RFP外,女性患者的耐药率均高于男性,耐多药率亦高于男性(P0.05)。结果提示,临床上应足量应用抗结核药物,对女性结核病患者要给予更多关注。     

结核分枝杆菌乙胺丁醇耐药机制的研究进展

耐多药结核病的出现和流行对结核病的防控造成了严重威胁。乙胺丁醇(Ethambutol, EMB)是一线抗结核药物,常与异烟肼、利福平等联合应用,还可用于耐药结核病的治疗。但近年来EMB耐药形势严峻,我国复治结核病患者中EMB耐药率已达17.2%,并呈上升趋势;耐多药结核病患者中,EMB耐药率约为51.3%~66.7%,情况不容乐观。明确EMB耐药的产生机制对于有效防控EMB耐药率的上升、充分发挥EMB的作用十分重要,因此本文对结核分枝杆菌EMB的耐药现状、EMB的作用机制及其耐药产生机制方面的研究进展进行了综述。     

结核分枝杆菌耐链霉素和乙胺丁醇分子机制的研究

目的:研究结核分枝杆菌耐链霉素和乙胺丁醇的rpsL和emb B基因突变情况,探讨耐药基因突变与耐药性的关系。方法:通过传统药敏实验和聚合酶链反应(PCR)--单链构象多态性(SSCP)技术初步鉴定62株临床分离株的药敏和rps L、emb B基因。结果:与结核菌标准株H37Rv对照,分析30例TB菌耐链霉素(SM)的rps L基因,发现其突变率为70.0%(21/30),分析29例耐乙胺丁醇(EMB)的emb B基因,该基因的突变率为65.5%(19/29)。结论:部分结核分枝杆菌耐SM和EMB是由于其rps L、emb B基因突变所致,PCR-SSCP银染技术可能成为测定部分结核分枝杆菌耐药的简便、快速的方法。     

应用基因芯片快速检测分枝杆菌

目的:将临床疑似分枝杆菌感染患者的标本用基因芯片方法进行分枝杆菌菌种鉴定,并将基因芯片检测的结果与传统的抗酸染色方法进行对比。方法:采用基因芯片PCR扩增、分子杂交、微阵列芯片扫描的方法检测379例临床疑似标本。结果:基因芯片阳性检出率为16.3%(62/379),涂片抗酸染色法阳性检出率为15.3%(58/379),两者差异无统计学意义(χ2=0.16,P>0.05);62例基因芯片检测阳性患者中有52例为结核分枝杆菌,另有10例为非结核分枝杆菌(其中3例偶然分枝杆菌,1例龟/脓肿分枝杆菌,1例堪萨斯分枝杆菌,4例浅黄分枝杆菌,1例海分枝杆菌)。结论:结合临床病例分析结果显示,基因芯片检测对鉴别结核分枝杆菌和非结核分枝杆菌分型具有快速、特异性高的特点,在分枝杆菌感染的早期诊断和治疗上具有重要的临床价值,值得推广和应用。     

城市生活饮用水中非结核分枝杆菌调查

目的 调查上海市生活饮用水中非结核分枝杆菌的状况及主要菌种分布。方法 应用过滤法收集细菌, 并在改良罗氏培养基上培养; 通过16S rRNA 测序鉴定菌种。结果 上海市生活饮用水中非结核分枝杆菌的检出率为16. 7% , 其中自来水厂原水、出厂水和居民生活饮用终端水的检出率分别为60% 、25% 和10. 3% 。分离鉴定的非结核分枝杆菌菌种为戈登分枝杆菌及偶发分枝杆菌, 分别占90% 及10% 。结论 在上海市居民生活饮用水系统中存在非结核分枝杆菌, 因此, 应当采取有效的控制方法以保护公众的健康。     

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...     

儿童结核病耐药状况及危险因素分析

目的了解最新儿童耐药结核病流行现状,为耐药儿童结核病的预防、控制提供依据。方法于2006年6月至2009年6月间收集了重庆医科大学附属儿童医院161例结核病患儿样本,采用比例法对链霉素(Streptomycin,SM)、异烟肼(Isoniazide,INH)、利福平(Rifampicin,RFP)、乙胺丁醇(Ethambutol,EMB)和吡嗪酰胺(Pyrazinamide,PZA)共5种药物进行耐药性测定。结果 161株菌株中鉴定出139例为结核分枝杆菌,对这139例培养阳性儿童结核病例中,总耐药率和总耐多药率分别为20.86%、6.47%,平均耐药率顺位由高到低是SM(12.2%)、INH(10.79%)、RFP(8.63%)、EMB(2.88%)、PZA(2.88%)。结论重庆地区儿童结核病总体耐药及耐多药水平低于全国平均水平,城镇地区及复治患儿可能是儿童结核病病例发生多耐药的相关因素。     

结核分枝杆菌eis 基因突变与氨基糖苷耐药的研究

目的:探讨结核分枝杆菌eis基因突变与氨基糖苷耐药之间的相互关系。方法:以本室保存的35株已确定耐一线药物(异烟肼、利福平、乙胺丁醇、链霉素)的结核分支杆菌为研究对象,应用BECTEC960测定其二线药物(阿米卡星、卡那霉素)的耐药情况,同时应用基因测序的方法测定结核分枝杆菌eis基因突变情况,分析eis基因突变与氨基糖苷耐药之间的相互关系。结果:氨基糖苷耐药的部分结核分杆杆菌中,eis基因487位碱基出现突变,相应的163位氨基酸密码子由CGT突变为CAT,即由缬氨酸变为异亮氨酸。结论:eis基因V163I突变(缬氨酸变为异亮氨酸)可能与结核分枝杆菌耐氨基糖苷类药物有关。     

Activities of Linezolid against nontuberculous mycobacteria

The activity of linezolid (Pfizer, USA) was tested by broth microdilution against 53 clinical isolates of non-tuberculous mycobacteria (NTM), including the common disease producing species Mycobacterium avium, M. intracellulare, M. fortuitum, M. chelonae and M. abscessus, obtained from western Turkey. The isolates of M. abscessus and M. intracellulare were the least susceptible, M. mucogenicum, M. gordonae and M. avium were the most susceptible to linezolid of the common species of NTM. Linezolid showed a variable sensitivity in all strains; therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before using the drug for human therapy.     

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center,Rio de Janeiro,Brazil, in pre highly active antiretroviral therapy era

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85% of the patients and NTM in 15%. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8% (49/83) of NTM isolates, most of them from sterile sites (75.5%), and in 94% (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99% of MAC are urease-negative), however IS1245 was detected in 96% of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2%), followed by M. terrae (3.6%), M. gordonae (2.4%), M. chelonae (1.2%), M. fortuitum (1.2%) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8%). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods.     

Evaluation of antimicrobial susceptibilities of rapidly growing mycobacteria by Sensititre RAPMYCO panel

This study used Sensititre RAPMYCO to test the activities of amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, sulfamehoxazole, tigecycline and tobramycin against 25 clinical isolates of rapidly growing mycobacteria (RGM), including the common disease producing species Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum and Mycobacterium peregrinum. Analysis of the four different RGM species showed that isolates of M. fortuitum and M. peregrinum were more susceptible than M. abscessus and M. chelonae. Different antimicrobials showed a variable sensitivity in all strains. Therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before the treatment of infections due to RGM.     

PCR with subsequent sequencing of the 16S gene rRNA in species identification of mycobacteria of the non-tuberculosis complex

One hundred of mycobacterium cultures were assayed by the method of PCR with subsequent sequencing of the 16S rRNA region. The below mycobacterium species were identified: M. tuberculosis complex (n = 55), M. avium (n = 17), M. intracellulare (n = 4), M. scrofaleceum (n = 2), M. kansasii - M. gastri (n = 3), M. gordonae (n = 3), M. ulcerans - M. marinum (n = 1), M. smegmatis (m = 2), M. fortuitum (n = 11), M. peregrinum (n = 1) and M. chelonae - M. abscessus (n = 1). The method enabled the differentiation of species M. avium from M. intracellulare and M. peregrinum from M. fortuitum, which could not be differentiated by using the classic biochemical and bacteriological methods. Genetic heterogeneity of the mycobacterium strains of M. avium, M. fortuitum and M. gordonae was also established by PCR plus sequencing of the 16S rRNA region.     

Heat susceptibility of aquatic mycobacteria.

An investigation was carried out to measure the heat susceptibility of opportunistic mycobacteria frequently isolated from domestic water supply systems. The study was conducted under standardized conditions designed to resemble those found in oligotrophic aquatic habitats. Strains of the following species were tested: Mycobacterium avium, M. chelonae, M. fortuitum, M. intracellulare, M. kansasii (two strains), M. marinum, M. phlei, M. scrofulaceum, and M. xenopi. Suspensions of the test strains were exposed to temperatures of 50, 55, 60, and 70 degrees C; samples were taken at defined intervals to determine the concentration of survivors. From these data, the decimal reduction times were calculated for each test strain and test temperature. The results indicate that M. kansasii is more susceptible to heat than Legionella pneumophila, whereas the heat susceptibilities of M. fortuitum, M. intracellulare, and M. marinum lie in the same order of magnitude as that of L. pneumophila. The strains of M. avium, M. chelonae, M. phlei, M. scrofulaceum, and M. xenopi were found to be more thermoresistant than L. pneumophila, with the highest resistance being found in M. xenopi. Thermal measures to control L. pneumophila may therefore not be sufficient to control the last five mycobacterial species in contaminated water systems.     

Drug resistance among Mycobacterium tuberculosis strains isolated in Taiwan during 1975.

455 strains of Mycobacterium tuberculosis were isolated from patients with history of treatment in Taiwan Provincial Tuberculosis Control Bureau and tested for resistance against various antituberculosis agents including streptomycin (SM), paraaminosalicylic acid (PAS), isoniazid (INH), cycloserine (CS), prothionamide (1321TH), kanamycin (KM), ethambutol (EMB), and rifampicin (RFP). In vitro resistance to SM and INH was more frequently found than others and the resistance to a single drug was more common than multiple resistance.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Expanding the mycobacterial diversity of metalworking fluids (MWFs): evidence showing MWF colonization by Mycobacterium abscessus

Nontuberculous mycobacteria (NTM) have been associated with hypersensitivity pneumonitis in machinists. Only two species of NTM, namely Mycobacterium immunogenum and Mycobacterium chelonae, have been reported thus far to have the ability to colonize contaminated metalworking fluids (MWFs). Here, we report, for the first time, the presence and characterization (phenotypic and genotypic) of a third species, Mycobacterium abscessus, colonizing these harsh alkaline machining fluids. Two Mycobacterium morphotypes, smooth (S) and rough (R), were isolated (two isolates each) from an in-use industrial MWFs. Biocide susceptibility analysis using triclosan as a model yielded the same minimal inhibitory concentration for the two morphotypes. PCR-restriction analysis-based speciation of the morphotypes confirmed their identity as M. abscessus. Genotyping based on partial DNA sequences corresponding to the variable regions of the hsp65 gene and 16S-23S rRNA operon internal transcribed spacer region and randomly amplified polymorphic DNA-PCR analysis showed that both morphotypes belong to a single genotype. In addition, we isolated and confirmed two novel mycobacterial genotypes, one each of M. immunogenum and M. chelonae from additional in-use MWF screening. Taken together, this study expands the known mycobacterial species- and strain-diversity colonizing MWF. Furthermore, the study emphasizes the need for including M. abscessus species in the existing mycobacterial screening of contaminated MWF.     

Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium

16S rRNA sequences from Mycobacterium tuberculosis, M. avium, M. gastri, M. kansasii, M. marinum, M. chelonae, M. smegmatis, M. terrae, M. gordonae, M. scrofulaceum, M. szulgai, M. intracellulare, M. nonchromogenicum, M. xenopi, M. malmoense, M. simiae, M. flavescens, M. fortuitum, and M. paratuberculosis were determined and compared. The sequence data were used to infer a phylogenetic tree, which provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. The groups of slow- and fast-growing mycobacteria could be differentiated as distinct entities. We found that M. simiae occupies phylogenetically an intermediate position between these two groups. The phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotchromogenic, and nonchromogenic mycobacteria. In general, the phylogenetic units identified by using rRNA sequences confirmed the validity of phenotypically defined species; an exception was M. gastri, which was indistinguishable from M. kansasii when this kind of analysis was used.     

Photochromogenic and scotochromogenic mycobacteria: their clinical significance

In the period 1973--1977, Mycobacterium tuberculosis was isolated by cultivation in 4408 cases from the clinical specimens of patients with positive X-ray findings. On the basis of atypical colony morphology or pigment formation, 263 other mycobacterial strains were identified: of these 23 were photochromogenic and belonged to Mycobacterium kansasii. The strains were cultured on several occasions from the specimens of 4 patients with broncho-pulmonary mycobacteriosis. The strains were resistant to isoniazid and streptomycin, sensitive to ethambutol and rifampicin. A total of 18 scotochromogenic isolates cultured from 14 patients with positive X-ray findings were identified as Mycobacterium aquae (M. gordonae) and its variants: strains showing slow Tween hydrolysis and 1 strain of rapid growth. In 5 cases M. tuberculosis was also obtained, indicating the presence of a mixed mycobacterial population. All scotochromogens were resistant to isoniazid and sensitive to ethambutol, with the exception of two strains sensitive to rifampicin.