Improved detection and differentiation of mycobacteria with combination of Mycobacterium Growth Indicator Tube and Roche COBAS AMPLICOR System in conjunction with Duplex PCR.

In this study, a combination of liquid and solid media (current "gold standard" for culture) with combinations of liquid media (Mycobacteria Growth Indicator Tube (MGIT)) plus a commercial amplification system (Roche COBAS AMPLICOR System (CAS)), and solid media (Ogawa) plus CAS for detection of Mycobacterium tuberculosis were compared. In addition, the ability of the MGIT to recover mycobacteria from various clinical samples was compared with the abilities of egg-based Ogawa medium using equal volume of samples and a high concentration (6%) of NaOH for decontamination. A total of 705 specimens (395 respiratory and 310 extrapulmonary) that were collected from 554 patients were tested in parallel with three assays. The results of MGIT and Ogawa were evaluated with the "gold standard" (combination of culture and clinical data) and those of CAS were evaluated with extended gold standard including treated tuberculosis. A total of 130 mycobacterial infections (M. tuberculosis, n=122; mycobacterium other than tuberculosis (MOTT), n=8) were detected. The differentiation of M. tuberculosis and MOTT was successfully accomplished using duplex PCR. The overall sensitivity of the MGIT, Ogawa, and CAS for M. tuberculosis was 89.9%, 73.9%, and 79.9%, respectively. For the MOTT, the corresponding values for the MGIT and Ogawa medium were 100% and 12.5%, respectively. The mean detection time for M. tuberculosis was 22 days using MGIT and 32 days when using the Ogawa medium. The specificity of CAS was 98.4%, with an inhibition rate of 1.4%. A combination of MGIT plus CAS detected 97.5% of all M. tuberculosis infections (compared with MGIT plus Ogawa, 91.8%, P<0.05; compared with Ogawa plus CAS, 87.7%. P<0.01). Our results indicate that a combination of MGIT plus a Roche CAS in conjunction with duplex PCR, would be quite useful in clinical laboratories for both rapid detection and differentiation of M. tuberculosis and MOTT.     

TRICORE, a novel bead-based specimen concentration method for the culturing of Mycobacterium tuberculosis

Centrifugation is a necessary concentrating step for the detection of Mycobacterium tuberculosis in a liquid culture. However, centrifugation is biologically hazardous and presents an obstacle in the development of an automated culture system. A bead-based bacterial concentration method, TRICORE, was recently developed by Genetein Co., Ltd. We compared the efficacy of TRICORE and conventional centrifugation for concentrating M. tuberculosis in clinical sputum specimens by using liquid and solid culture systems. Among 90 pretreated clinical sputum specimens, 51 (57.3%) and 55 (61.8%) M. tuberculosis isolates were recovered by the MGIT culture system by using the centrifugation and TRICORE methods, respectively (chi-square test, p=0.5413). The detection time for the centrifugation method was 359.3±117.0 h, while that for the bead-based concentration method was 377.6±162.3 h (p=0.5637). However, the number of colonies recovered on solid media were significantly higher with the TRICORE method (p=0.003). In particular, among the smear-negative specimens, culture positivity of the TRICORE method was 39.6%, while that of the centrifugation method was 15.1%. The TRICORE bead-based concentration method was considered equivalent to centrifugation and enabled efficient collection of paucibacillary specimens in solution. Thus, the new noncentrifugation concentration method could yield more positive culture results.     

复治肺结核患者结核分枝杆菌L型培养结果分析

目的：了解复治肺结核患者的结核分枝杆菌L型培养情况,探讨结核分枝杆菌L型阳性与耐多药的关系。方法：选择180例肺结核患者的痰标本进行结核分枝杆菌培养和结核分枝杆菌L型培养,同时对110例复治组中培养阳性的标本行耐药监测。结果：复治组的L型阳性率为43．6％,初治组的L型阳性率为15．7％,复治组显著高于初治组（P〈0．01）;菌阳复治组的L型阳性率50％,菌阴复治组的L型阳性率39．4％,菌阳组明显高于菌阴组（P〈0．05）;L型菌阳性患者的耐药率显著高于L型菌阴性组（P〈0．05）。结论：结核分枝杆菌L型阳性是引起结核病复发、耐药的重要原因;MDR-TB与结核分枝杆菌L型感染有关。     

Comparison of the mycobacteria growth indicator tube with radiometric and solid culture for isolation of mycobacteria from clinical specimens and susceptibility testing of Mycobacterium tuberculosis

We compared the mycobacteria growth indicator tube (MGIT) system with the BACTEC 460 TB and Loewenstein-Jensen (LJ) systems for the recovery of mycobacteria (acid-fast bacilli [AFB]) from 600 clinical specimens. A total of 50 AFB (32 Mycobacterium tuberculosis complex, 10 M. avium complex, 3 M. gordonae, 3 M. xenopi, 1 M. terrae and 1 M. fortuitum) were detected. MGIT recovered 50 isolates of AFB (100% sensitivity), and BACTEC 460 TB and LJ recovered 49 (98% sensitivity) and 19 (38% sensitivity) AFB isolates, respectively. The mean times to detect mycobacteria were 10, 10 and 25 days for MGIT, BACTEC 460, and LJ slants. All isolates of M. tuberculosis complex were tested for susceptibility to streptomycin, isoniazid, rifampin, and ethambutol with the MGIT and BACTEC 460 TB. Both systems yielded identical susceptibility data with different mean times to report (5.38 days for MGIT versus 7.33 days for BACTEC 460 TB, P<0.05). The results suggest that MGIT is equivalent to BACTEC 460 TB in its ability to support the growth of mycobacteria, but significantly more efficient than LJ. MGIT may also be used for susceptibility testing of primary antituberculosis drugs.     

Xpert MTB/RIF在人类免疫缺陷病毒感染/艾滋病患者中诊断结核病的价值

为探讨利福平耐药结核分枝杆菌实时荧光定量核酸扩增检测技术(Xpert Mycobacterium tuberculosis/rifampicin,Xpert MTB/RIF)在人类免疫缺陷病毒感染/艾滋病(human immunodeficiency virus infection/acquired immunodeficiency syndrome,HIV/AIDS)患者中诊断结核病的价值,本研究回顾性分析了2018年1月1日—2020年12月31日复旦大学附属公共卫生临床中心感染与免疫科收治的801例HIV/AIDS合并疑似结核病患者的临床资料。801例患者中,657例进行了Xpert MTB/RIF、外周血结核感染T细胞斑点试验(tuberculosis T cell spot test,T-SPOT.TB)、抗酸染色涂片镜检和BACTEC MGIT 960液体培养等检测。以液体培养及菌型鉴定结果作为结核病诊断的“金标准”,确诊结核病92例,Xpert MTB/RIF、T-SPOT.TB、抗酸染色涂片镜检在HIV/AIDS患者中诊断结核病(包括肺结核和肺外结核)的灵敏度分别为72.8%、55.4%和69.6%,特异度分别为96.8%、90.3%和84.4%,与“金标准”行一致性检验,Kappa值分别为0.719 (P<0.01)、0.430(P<0.01)和0.424(P<0.01)。Xpert MTB/RIF检测502份呼吸道样本,结果显示其诊断肺结核的灵敏度和特异度分别为66.7%和96.0%;在痰涂片阳性和阴性的患者中,Xpert MTB/RIF诊断肺结核的灵敏度分别为77.4%和35.2%,特异度分别为87.7%和 97.8%。采用Xpert MTB/RIF检测343份肺外标本,结果显示其诊断肺外结核的灵敏度和特异度分别为63.3%和95.2%。以上结果提示,Xpert MTB/RIF在HIV/AIDS患者中诊断结核病(包括肺结核和肺外结核)具有较高的灵敏度和特异度,诊断肺结核的灵敏度高于肺外结核,因此推荐将其作为HIV/AIDS患者疑似结核病的首选检测方法。     

Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. In respect to high GC content of Mycobacterium tuberculosis, nonsynonymous mutations are dominant in this group. In this study a collection of 145 M. tuberculosis isolates was used to evaluate the conferring mutations in nucleotide 1388 of katG gene (KatG463) in resistance to isoniazid. A PCR-RFLP method was applied in comparison with DNA sequencing and anti-mycobacterial susceptibility testing. From all studied patients, 98 (67.6%) were men, 47 (32.4%) were women, 3% were <15 and 9% were >65 years old; male to female ratio was 1:2.4. PCR result of katG for a 620-bp amplicon was successful for all purified M. tuberculosis isolates and there was no positive M. tuberculosis culture with PCR negative results (100% specificity). Subsequent PCR RFLP of the katG identified mutation at KatG463 in 33.3%, 57.8% and 59.2% of our clinically susceptible, multidrug resistant TB (MDR) and extensively drug resistant (XDR) isolates, respectively. Strains of H37Rv and Academic had no any mutations in this codon. M. bovis was used as a positive control for mutation in KatG463. Automated DNA sequencing of the katG amplicon from randomly selected INH-susceptible and resistant isolates verified 100% sequence accuracy of the point mutations detected by PCR-RFLP. We concluded that codon 463 was a polymorphic site that is associated to INH resistance (a missense or "quiet" mutation). RFLP results of katG amplicons were identical to those of sequence method. Our PCR-RFLP method has a potential application for rapid diagnosis of M. tuberculosis with a high specificity.     

Bacteriological follow-up of pulmonary tuberculosis treatment: a study with a simple colorimetric assay

The viability of Mycobacterium tuberculosis (MTB) in serial sputum specimens from persistently smear positive patients was evaluated. The assay was based on oxidation-reduction of Alamar Blue and Malachite Green dyes that change their color in response to MTB growth. A total of 280 sputum specimens from 40 persistently smear positive TB patients and 40 sputa from non-tuberculosis patients were digested, decontaminated and examined microscopically. To check the MTB viability, the sediments from decontaminated samples were inoculated into three culture media: Lowenstein-Jensen (LJ) slants, Alamar Blue and Malachite Green culture tubes. We found that out of 280 smear positive specimens, the LJ culture was positive in 124 (44%). The numbers of correctly identified S+/C+ cases by Alamar Blue and Malachite Green were 118 (95%) and 116 (93%), respectively. The mean time required for reporting the positive signal in Alamar Blue culture tubes was 9 versus 11 days by Malachite Green culture tubes. In the standard LJ culture media the average detection time was 27 days (P < 0.05). The sensitivity of LJ was 99%, Alamar Blue 95% and Malachite Green 93%. The specificity was 100%, 92% and 93%, respectively. The oxidation-reduction method is rapid, sensitive and inexpensive in monitoring the treatment response of patients with pulmonary TB. Thus, using this method can be of paramount importance, particularly in resource-constrained areas.     

Identification of Mycobacterium tuberculosis by PCR-linked reverse hybridization using specific rpoB oligonucleotide probes

A reverse probe hybridization method using two different Mycobacterium tuberculosis-specific rpoB DNA probes in combination was evaluated for the identification of M. tuberculosis culture isolates. Among the 384 isolates tested, 354 strains were identified as M. tuberculosis, which included 37 rifampin-resistant strains, and 30 were nontuberculous mycobacteria (NTM). This result was in accord with partial rpoB sequence analysis and IS6110 polymerase chain reaction (PCR) results, but not with the results of biochemical testing, which produced two false negative results. Because of its high level of sensitivity and specificity, we suggest that M. tuberculosis-specific rpoB probes immobilized on micro-titer well plates or on other solid matrixes can be used efficiently for the rapid and convenient identification of M. tuberculosis.     

Molecular identification and characterization of rifampicin-resistant Mycobacterium tuberculosis isolates by line probe assay: an approach for rapid diagnosis of multidrug-resistant tuberculosis

Aim:  Early identification and characterization of rifampicin-resistant (R^r) Mycobacterium tuberculosis isolates recovered from the samples of tuberculosis (TB) patients in the Aegean (West Anatolian) Region was intended.
Methods and Results:  Sixty isolates [47 (78·3%) multidrug-resistant (MDR)], which were identified as M. tuberculosis complex and phenotypically resistant to rifampicin by both BACTEC mycobacteria growth indicator tube (MGIT) 960 and 460 systems were analysed by a commercial line probe assay (INNO-LiPA Rif TB). The concordance of LiPA with the in vitro susceptibility test was found as 98·3%. Among the isolates, S531L (R5 pattern; 46·7%) and L511P/R, S512T, Q513L/K (ΔS1 pattern; 11·7%) were the most frequent mutation patterns. As compared with the BACTEC systems and conventional techniques for cultivation, identification and in vitro susceptibility testing, INNO-LiPA Rif TB after cultivation in BACTEC MGIT 960 system provided an average of 20 days early diagnosis of R^r M. tuberculosis isolates.
Conclusions:  Rapid molecular identification and characterization of R^r M. tuberculosis isolates after BACTEC MGIT 960 cultivation would be useful for faster diagnosis, infection control and planning of accurate treatment in MDR-TB patients.
Significance and Impact of the Study:  Patients with MDR-TB need a specified treatment and efficient follow-up strategies. Rapid and practical methodologies to diagnose and follow these patients should be applied in routine use.     

Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.     

Rapid and simple approach for identification of Mycobacterium tuberculosis and M. bovis by detection of regulatory gene whiB7

Identification of Mycobacterium tuberculosis and M. bovis is necessary for the application of adequate drug therapy. PCR amplification is a good tool for this purpose, but choosing proper target is of a great concern. We describe a PCR assay for fast detection of M. tuberculosis and M. bovis.As a BLAST and BLASTP search we selected regulatory gene whiB7 that encodes multi-drug resistance in this bacterium. Thirty clinical isolates of M. tuberculosis were sequenced and all the mutations in gene whiB7 were detected. The best set of several pairs of primers was selected and used in comparison by rpoB gene for differentiation of M. bovis, M. avium, M. kansasii, M. phlei, M. fortuitum, M. terrae, seven non-pathogenic Mycobacterium isolates and 30 clinical isolates of M. tuberculosis.It was proved that only clinical isolates of M. tuberculosis and M. bovis have positive bands of 667 bp whiB7. Other non-tuberculous and non-pathogenic isolates did not show any positive sign. Furthermore, 667-bp PCR products of whiB7 gene were observed for ten positive sputum samples (preliminarily approved to be positive for M. tuberculosis by commercially real-time based method), but no bands were detected in 5 negative sputum samples. RpoB gene could not differentiate non-tuberculous strains and non-pathogenic isolates from pathogenic clinical isolates. We concluded that PCR amplification of the gene coding for the WhiB7 protein could be successfully used as a good tool for rapid identification of M. tuberculosis and M. bovis. We propose application of this method as a rapid and simple approach in mycobacteriological laboratories.     

An efficient alternative marker for specific identification of Mycobacterium tuberculosis

Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers.     

聚合酶链式反应检测结核杆菌的研究

以人型结核杆菌基因组DNA为模板,合成二段引物各20个碱基进行聚合酶链式反应(PCR)。经琼脂糖凝胶电泳证实,获得一条245bp扩增带。PCR检测的敏感性染色体基因组DNA为1pg,菌悬液为13个活菌／ml。在特异性试验中,人型结核杆趋,牛型结核杆菌、BCG可见此扩增带。被试的其它14种扰酸菌以及变铅青链霉菌、大肠杆菌质粒Puc19、星状诺卡氏菌、红球菌均未见该扩增带。54例肺结核痰标本3种方法检查的阳性率分别为：萋尼氏抗酸染色16．7％,培养法14．8％,PCR 37．0％。前2种检查方法分别与PCR比较,经统计学处理均有显著性差异(P<0．01)。12例非结核性肺部疾患痰标本抗酸染色和PCR均为阴性。结果表明,PCR技术是快速、敏感、特异诊断结核病的方法。     

Cost-effectiveness of blood agar for isolation of mycobacteria

Background

Mycobacterium species are grown using specific media that increase laboratory cost, thus hampering their diffusion in resource-limited countries. Preliminary data suggested that versatile blood agar may be also used for mycobacterial culture.

Methodology

We examined the growth of 41 different Mycobacterium species on 5% blood agar. Over a 24-month period we analysed isolation of mycobacteria after parallel inoculation of clinical specimens into both a reference automated system (BACTEC 9000 MB broth) and 5% blood agar slant tubes, after NaOH decontamination, and compared the cost of performing 1,000 analyses using these two techniques.

Conclusions

Mycobacterium reference species cultured on blood agar, with the exception of Mycobacterium ulcerans. Inoculation of 1,634 specimens yielded 95 Mycobacterium isolates. Blood agar performed significantly more efficiently than BACTEC 9000 MB broth (94 vs 88 isolates, P = 0.03). Decontamination of Candida albicans in 5 specimens by addition of amphotericin B in blood agar yielded one more M. tuberculosis isolate that could not be isolated in BACTEC broth. Uneven distribution of time to culture positivity for M. tuberculosis had a median (range) of 19±5 days using blood agar and 26±6 days using BACTEC 9000 MB broth. Cost for 1,000 analyses in France was estimated to be of 1,913 euros using the blood agar method and 8,990 euros using the BACTEC 9000 MB method. Blood agar should be regarded as a first-line medium for culturing Mycobacterium species. It saves time, is cost-effective, is more sensitive than, and at least as rapid as the automated method. This is of particular importance for resource-limited countries in which the prevalence of tuberculosis is high.     

Rapid mycobacteria drug susceptibility testing using Gel Microdrop (GMD) Growth Assay and flow cytometry

Control of multi-drug-resistant tuberculosis has been hampered by the lack of simple, rapid and sensitive methods for assessing bacterial growth and antimicrobial susceptibility. Due to the increasing incidence and high frequency of mutations, it is unlikely that culture methods will disappear in the foreseeable future. Therefore, the need to modernize methods for rapid detection of viable clinical isolates, at a minimum as a gold standard, will persist. Previously, we confirmed the feasibility of using the Gel Microdrop (GMD) Growth Assay for identifying sub-populations of resistant Mycobacteria by testing different laboratory strains. Briefly, this assay format relies on encapsulating single bacterium in agarose microspheres and identifying clonogenic growth using flow cytometry and fluorescent staining. In this study, we modified the GMD Growth Assay to make it suitable for clinical applications. We demonstrated the effectiveness and safety of this novel approach for detecting drug susceptibility in clinically relevant laboratory strains as well as clinical isolates of Mycobacterium tuberculosis. Correlation between results using the GMD Growth Assay format and results using two well characterized methods (Broth Microdilution MIC and BACTEC 460TB) was 87.5% and 90%, respectively. However, due to the inherent sensitivity of flow cytometry and the ability to detect small (<1%) sub-populations of resistant mycobacteria, the GMD Growth Assay identified more cases of drug resistance. Using 4 clinically relevant mycobacterial strains, we assessed susceptibility to primary anti-tuberculosis drugs using both the Broth Microdilution MIC method and the GMD Growth Assay. We performed 24 tests on isoniazid-resistant BCG, Mycobacterium tuberculosis H37Ra and Mycobacterium avium strains. The Broth Microdilution MIC method identified 7 cases (29.1%) of resistance to INH and EMB compared to the GMD Growth Assay which identified resistance in 10 cases (41.6%); in 3 cases (12.5%), resistance to INH and EMB was detected only with the GMD Growth Assay. In addition, using 20 Mycobacterium tuberculosis clinical isolates, we compared results using BACTEC 460TB method performed by collaborators and the GMD Growth Assay. Eight of 20 (40%) clinical isolates, which were not identified as drug-resistant using the conventional BACTEC 460TB method, were resistant to 1, 2, or 3 different concentrations of drugs using the GMD Growth Assay (13 cases of 140 experiments). In one case (isolate 1879), resistance to 10.0 microg/ml of STR detected using BACTEC 460TB method was not confirmed by the GMD Growth Assay. Thus, the overall agreement between these methods was 90% (14 discrepant results of 140 experiments). These data demonstrate that the GMD Growth Assay is an accurate and sensitive method for rapid susceptibility testing of Mycobacterium tuberculosis for use in clinical reference laboratory settings.     

Standardization of in-house polymerase chain reaction for the identification of Mycobacterium tuberculosis at the reference Tropical Disease Hospital in the State of Goiás, Brazil

This study compares smear, growth in Lowenstein-Jensen medium, and in-house polymerase chain reaction (PCR) techniques for the detection of Mycobacterium tuberculosis. A total of 72 specimens from 72 patients with clinical symptoms of tuberculosis, including 70 sputum and two bronchial aspirate samples, were tested in parallel by smear, culture, and in-house PCR techniques. From these, 48 (66.6%) were negative by the 3 methods, 2 (2.8%) were smear positive and negative by culture and in-house PCR, 11 (15.3%) were both smear and culture negative, and in-house PCR positive, 7 (9.7%) were positive by the 3 methods, 2 (2.8%) were positive by smear and culture, and negative by PCR, 2 (2.8%) were positive by culture and PCR, but smear negative. After the resolution of discrepancies in PCR results, the sensitivity and specificity for in-house PCR technique to M. tuberculosis relative to the culture, were 81.8% and 81.9%, respectively. These results confirm that this method, in-house PCR, may be a sensitive and specific technique for M. tuberculosis detection, occurring in both positive and negative smear and negative cultures.     

微菌落观察法快速检测和鉴别结核杆菌培养物的研究

将取自肺结核患者的219例痰标本接种匡氏琼脂平板,共分离到112例结核菌生长物。其中培养法104例(92.8％)阳性,微菌落观察法108例(96.4％)阳性,微菌落法阳性率稍高。培养法和微菌落法阳性标本首次检出时间分别为18.6d和11d,微菌落检出时间更短(P<0.01)。常规菌型鉴别方法与微菌落法对结核杆菌菌型鉴别的符合率为99％。     

Cytochrome CYP141: a new target for direct detection of Mycobacterium tuberculosis from clinical specimens

Cytochrome P450 CYP141 is an intermediary metabolic and respiratory protein that interferes with oxidation reduction in Mycobacterium tuberculosis. This conserved protein has also been debated as a hypothetical target for therapeutics. We used the sequences of CYP141 gene to develop a PCR for rapid detection of Mycobacterium tuberculosis from respiratory specimens. The sensitivity of this PCR for culture positive-smear positive and culture positive-smear negative samples were 92% and 62.5%, respectively. The overall sensitivity and specificity of this PCR was 85.7% and 97.8%. As compared with other studies, it appears that the CYP141 gene is a good target for direct detection of M. tuberculosis from respiratory specimens.     

Usefulness of the BACTEC MYCO/F lytic system for detection of mycobacteremia in a clinical microbiology laboratory

278 BACTEC MYCO/F lytic system blood cultures for mycobacteria were evaluated between 1997 and 1999. Sixty of them were read as positive by the system, being considered 15 of them as false positives. Twenty-seven yielded mycobacterial growth (13 Mycobacterium avium from 3 patients and 14 Mycobacterium tuberculosis from 8 patients). Other bacteria isolated were coagulase-negative Staphylococcus (13 samples), Corynebacterium sp. (5 samples), Salmonella enteritidis (2 samples) and Klebsiella pneumoniae (1 sample). Five of these isolates were considered as true episodes of bacteremia. The average time for detection of mycobacteria was 12.6 days for M. avium and 26.4 days for M. tuberculosis. BACTEC MYCO/F lytic system is useful for detection of mycobacteremia in clinical microbiology laboratories.     

一种随机引物联合多特异性DNA片段检测结核分支杆菌的新方法

目的:针对目前结核性疾病实验室诊断的局限性,探索一种更为敏感和特异的结核分枝杆菌DNA检测新方法。方法:选取10株江苏地区流行的结核分支杆菌(MTB)菌株,选取临床其他常见菌株及分枝杆菌菌株作为对照组,分别提取DNA作为随机引物的模板。参考国内、外文献设计12条随机引物,并分别对MTB及对照菌株进行单个引物随机扩增,2%的琼脂糖凝胶电泳对扩增产物进行分离并切胶纯化,通过TA克隆将纯化片段连接到质粒pEASYTM-T5 Zero并进行测序,通过BLAST-nr比对验证是否为MTB DNA片段。按照所确定的MTB片段序列,在其内部设计、合成一对特异性引物。用此特异性引物扩增对应的随机引物扩增产物,获得MTB特异性条带图谱。并将该方法检测的敏感性和特异性与临床上常用的real-time PCR进行比较。结果:经BLAST-nr比对,随机引物IS986F,S535及IS986R扩增的条带与MTB DNA有高度同源性(均为99%)。随机引物IS986F、S535和IS986R分别联合其特异性引物可以检测稀释105倍、105倍和103倍的MTB DNA,其特异性分别为100%、90%和80%。常规real-time PCR可检测出稀释104倍的MTB DNA。结论:随机引物IS986F联合其特异性引物检测结核分枝杆菌的灵敏度和特异性优于S535、IS986R两组,特异性为100%,且灵敏度优于常规real-time PCR法。