Molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates from Shanghai, China

China is one of the countries with the highest prevalence of fluoroquinolone-resistant (FQ(r)) Mycobacterium tuberculosis. Nevertheless, knowledge on the molecular characterization of the FQ(r)M. tuberculosis strains of this region remains very limited. This study was performed to investigate the frequencies and types of mutations present in FQ(r)M. tuberculosis clinical isolates collected in Shanghai, China. A total of 206 FQ(r)M. tuberculosis strains and 21 ofloxacin-sensitive (FQ(s)) M. tuberculosis strains were isolated from patients with pulmonary tuberculosis in Shanghai. The phenotypic drug susceptibilities were determined by the proportion method, and the mutations inside quinolone resistance-determining region (QRDR) of gyrA and gyrB genes were identified by DNA sequence analyses. Among 206 FQ(r)M. tuberculosis strains, 44% (90/206) were multidrug-resistant isolates and 39% (81/206) were extensively drug-resistant isolates. Only 9% (19/206) were monoresistant to ofloxacin. In total, 79.1% (163/206) of FQ(r) isolates harboured mutations in either gyrA or gyrB QRDR. Mutations in gyrA QRDR were found in 75.7% (156/206) of FQ(r) clinical isolates. Among those gyrA mutants, a majority (75.6%) harboured mutations at amino acid position 94, with D94G being the most frequent amino acid substitution. Mutations in gyrA QRDR showed 100% positive predictive value for FQ(r)M. tuberculosis in China. Mutations in gyrB were observed in 15.5% (32/206) of FQ(r) clinical isolates. Ten novel mutations were identified in gyrB. However, most of them also harboured mutations in gyrA, limiting their contribution to FQ(r) resistance in M. tuberculosis. Our findings indicated that, similar to other geographic regions, mutations in gyrA were shown to be the major mechanism of FQ(r) resistance in M. tuberculosis isolates. The mutations in gyrA QRDR can be a good molecular surrogate marker for detecting FQ(r)M. tuberculosis in China.     

Dynamics of the laboratory results in patients with pulmonary tuberculosis

The process of infection and disease development is difficult to follow-up before tuberculosis (TB) confirmation. The laboratory analysis mirrors the infection with the possible subsequent breakdown to clinical TB. To better define the dynamics of the laboratory results in suspected and already confirmed patients with pulmonary TB, we studied the analysis of 1467 pathologic samples collected during the hospitalization of 841 patients. The samples were analyzed by 3 laboratory methods—direct microscopy, culture, and polymerase chain reaction (PCR). It was found that compared to cultures, the PCR method is more sensitive. For few cases, we demonstrate that the PCR result is positive about 2 weeks before the first positive culture. During the treatment follow-up, the PCR result remains positive for a long time, up to 4 to 5 months after the last positive culture. For better definition of the period during which microscopy and culture results remain positive, we studied the laboratory results of 100 casually selected patients with pulmonary TB positive on culture. The median periods during which these patients were found to be microscopy and culture positive were 10 and 25 days, respectively. Second to the dynamics of the laboratory results, we demonstrate that TB development is very rapid, whereas the period of recovery is long. The PCR results have to be reproducibly negative to accept that the process of active therapy is completed and the patient can remain under surveillance. On the basis of the laboratory data obtained, we propose empiric models for the dynamics of the laboratory results for patients with pulmonary tuberculosis.     

Clinical significance of Pneumocystis jiroveci in patients with active tuberculosis

Pneumocystis colonization has been associated with airway inflammation and obstruction. We conducted a retrospective cohort study to investigate the clinical significance of Pneumocystis in the airway of patients with active tuberculosis. Of the 108 respiratory specimens tested positive for M. tuberculosis by polymerase chain reaction (PCR), 11 (10.2%) were also positive for Pneumocystis by PCR. Compared with patients tested negative for Pneumocystis, those with Pneumocystis had a higher serum alanine transaminase level, a greater likelihood of requiring oxygen supplementation, and a worse 30-day mortality. The proportion of patients with chronic obstructive pulmonary disease was not significantly different between the 2 groups, but lung malignancy was more prevalent among patients with Pneumocystis. Multivariate analysis showed that Pneumocystis was independently associated with oxygen supplementation. Our study has shown an association between the detection of Pneumocystis in lower respiratory tract specimens and greater impairment of pulmonary function among patients with active tuberculosis.     

Epidemiologic surveillance to detect false-positive Mycobacterium tuberculosis cultures

This study was aimed to investigate the ability of potential indices from epidemiologic surveillance to detect false-positive cultures of Mycobacterium tuberculosis (MTB). All clinical specimens for mycobacterial culture from April 1 to August 31, 2010, were reviewed. Single-positive cultures without relevant clinical and pathologic information were categorized as suspected false-positive cultures. Genotyping methods were used to confirm false-positive cultures. The performance of epidemiologic surveillance indices to detect potential false-positive cultures was evaluated. A total of 14,462 specimens were sent to the laboratory and 214 batches were processed in 107 work days (average 67.6 specimens per batch, ranging from 21 to 130 specimens per batch). Seventy-one single-positive cultures were identified, among which 5 cultures of multidrug-resistant MTB in 1 batch were false-positive, confirmed by genotyping methods. Epidemiologic surveillance with statistical process control charts for single-positive cultures per day showed good performance in epidemiologic surveillance. The false-positive rate was 38.5% in the 13 potential false-positive cultures according to the statistical process control chart for single-positive cultures per day. Although the incidence of tuberculous disease is high in Taiwan, clustering of multidrug-resistant MTB in 1 batch or clustering of single-positive cultures still suggested the occurrence of false-positive MTB cultures. Therefore, epidemiologic surveillance for the clustering of single-positive cultures with the statistical process control chart could be used to monitor the occurrence of false-positive results.     

Evaluation of an immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in clinical isolates

Identification of Mycobacterium tuberculosis complex (MTC) remains slow. Over the years, several new technologies have been proposed to accelerate and simplify the detection of MTC. In this context, we evaluated an immunochromatographic assay (ICA) (BIO-LINE SD Ag MPT64 TB) for rapid identification of MTC, based on detection of a specific MPT64 antigen of MTC. We have tested it on i) mycobacterial cultures: 210 MTC strains and 28 nontuberculous mycobacteria; ii) M. bovis bacille Calmette-Guérin strain SSI (Statens Serum Institut, Denmark); and iii) 22 microorganisms other than mycobacteria, isolated from cultures. We concluded that this kit has an excellent specificity (100%) and sensitivity (99%) from isolated cultures. The ICA (BIO-LINE SD Ag MPT64 TB) allows excellent MTC identification from clinical isolates. It is a rapid, simple, and inexpensive test, and has a definite contribution in the rapid laboratory diagnosis of tuberculosis.     

Validation of the agar proportion and 2 liquid systems for testing the susceptibility of Mycobacterium tuberculosis to moxifloxacin

Moxifloxacin (MOX), an 8-methoxyquinolone compound, is now widely used for the treatment of bacterial infections and also accepted as 2nd-line drug for the treatment of multidrug-resistant (MDR) tuberculosis. To tentatively correlate the clinical outcome with in vitro results, we sought to set up susceptibility test conditions for Mycobacterium tuberculosis against MOX by using the reference agar proportion method, the BACTEC 460 radiometric system, and the recently validated nonradiometric BACTEC MGIT 960 system. Our aim was to determine the critical MOX test concentration to be used with the abovementioned methods for routine susceptibility testing. MICs were determined for 20 pan-susceptible strains, 10 MDR strains, and 10 fluoroquinolone-resistant strains with defined gyrA mutations. MOX MICs resulted in a bimodal pattern with values for gyrA mutants considerably higher than those for pan-susceptible and MDR strains. Our data showed that a concentration of 0.5 microg/mL allowed a clear-cut separation between susceptible and resistant strains when tested by all the studied methods. Confirmatory test with a subset of pan-susceptible and MDR isolates appeared to validate the selected critical concentration. The MOX-resistant strains were almost isolated from patients with prior fluoroquinolone exposure.     

A review on various heterocyclic moieties and their antitubercular activity

Tuberculosis (TB), a contagious infection caused by Mycobacterium tuberculosis, still remains the leading cause of the worldwide death among the infectious disease. Different moieties like pyrazoline, benzimidazol, purines, thiazole, flouroquinolones, quinoxaline, oxadiazol, pyrazol, thiozolidinones and azetidinones have been studied, synthesized and evaluated worldwide against M. tuberculosis to show their antitubercular activity.     

Expression analysis of efflux pump genes among drug-susceptible and multidrug-resistant Mycobacterium tuberculosis clinical isolates and reference strains

Finding a gene or genes that are involved with multidrug resistance will be useful for finding a new target for the treatment of drug resistant tuberculosis. In this study, we aimed to compare the differences of the expression of 15 putative multidrug efflux pump genes in clinically isolated drug sensitive and multidrug resistant (MDR) Mycobacterium tuberculosis isolates, and reference strains. We found that these genes in the drug-sensitive and MDR M. tuberculosis isolates have similar rates of expressions. However, we found the expression levels of the all the genes are significantly higher in the clinical strains compared to the expression level of genes in the reference strains. In addition to this, it is found that standard strain has lower MIC value for the drugs including streptomycin and rifampin compared to the clinical isolate. We presume that the increase of the gene expression in the clinical strains is due to the exposure of antituberculosis drugs during treatment of patients, which cause constitutive expression of efflux systems, which might increase MIC levels of the major anti-tuberculosis drugs.     

Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.     

T-cell responses to the Mycobacterium tuberculosis-specific antigens in active tuberculosis patients at the beginning,during, and after antituberculosis treatment

The objectives of the study were to assess the performance of the QuantiFERON-TB Gold In-Tube (QFN-G-IT) and the T-SPOT.TB tests in the immunodiagnosis of active tuberculosis (TB) in adult patients, and to study the T-cell interferon γ (IFN-γ) responses during treatment and in patients who have recovered after curative treatment and self-healed TB patients. When only analyzing patients included at the beginning of treatment, the sensitivity was 83.3% for T-SPOT.TB and 69.4% for QFN-G-IT. In contrast, when evaluating patients during treatment, the sensitivity of the T-SPOT.TB and QFN-G-IT decreased to 69.8% and 48.8%, respectively. The response to the specific antigens increased after finishing the treatment compared with the values during the treatment. The T-SPOT.TB was more sensitive in diagnosing active TB than the QFN-G-IT. The IFN-γ tests could be used as a complementary method in the diagnosis of active TB.     

Standardization and evaluation of a tetraplex polymerase chain reaction to detect and differentiate Mycobacterium tuberculosis complex and nontuberculous Mycobacteria--a retrospective study on pulmonary TB patients

The clinical presentation of pulmonary tuberculosis by members of Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) cannot be differentiated using the available standard diagnostic procedures. A single-tube tetraplex polymerase chain reaction (T-PCR) was designed to simultaneously amplify 4 well-known DNA targets of MTC. Taguchi's protocol was followed for the optimization of the conditions and was then tested on 288 pulmonary TB patient samples. The analytical sensitivity of the T-PCR was 100 fg of purified mycobacterial DNA, and specificity was found to be 100% in being able to distinguish MTC and NTM in all the cases tested. The results correlated well when validated with hsp65 PCR restriction analysis and sequencing of the 16S-23S internal transcribed spacer region, hsp65, and rpoB. The T-PCR described here is a quick, valuable, and cost-effective tool for determining whether the causative organism is MTC or NTM, and thus is useful for disease surveillance.     

耐吡嗪酰胺结核分枝杆菌基因突变研究

目的 研究吡嗪酰胺酶编码基因pncA突变与结核分枝杆菌吡嗪酰胺(pyrazinamide,PZA)耐药的关系.方法 将临床分离的36株结核分枝杆菌进行常规PZA药敏试验和pncA基因序列分析.采用绝对浓度法进行PZA药敏试验;PCR扩增pncA基因及其上游、下游碱基序列,纯化回收PCR产物克隆到T载体并进行全自动测序.结果 36株临床分离株中25株PZA耐药,11株PZA敏感.5株高耐PZA(PZA浓度为250 μg/ml)临床分离株4株pncA基因突变;20株低耐PZA(PZA浓度为50 μg/ml)6株pncA基因突变;25株耐PZA结核分枝杆菌pncA基因突变率为40.0%.3株PZA敏感临床分离株pncA基因出现突变、其中2株为同义突变.11株耐PZA结核分枝杆菌pncA基因上游调控序列突变,其中5株同时有pncA基因突变;2株PZA敏感结核分枝杆菌pncA基因上游序列突变.结论 pncA基因突变可引起结核分枝杆菌对PZA耐药,但pncA基因突变只是结核分枝杆菌耐PZA的一种机制,可能还存在PZA的其他耐药机制.     

Mycobacterium tuberculosis rrs A1401G mutation correlates with high-level resistance to kanamycin,amikacin, and capreomycin in clinical isolates from mainland China

Mutations correlating phenotypic resistance level with the injectable second-line anti-tuberculosis drugs (SLDs) including kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) remain elusive. A collection of 114 Mycobacterium tuberculosis clinical isolates from mainland China was analyzed. The minimum inhibitory concentration (MIC) of each strain was determined and the sequences of rrs, tlyA, promoter of eis as well as 5′ untranslated region (UTR) of whiB7 were amplified and sequenced. No mutation in tlyA, promoter of eis and 5′ UTR of whiB7, was found to be associated with resistance among these samples. Sequencing data of 1400 rrs region demonstrated the A1401G mutation in rrs was prevalent, which presented in 84% of the KAN resistant isolates while only in about 50% of the AMK or CAP resistant isolates. Furthermore, most of the resistant isolates with A1401G mutation showed high-level resistance to these injectable SLDs. In conclusion, our results suggest the rrs A1401G mutation was related to high-level resistance to KAN, AMK, and CAP in M. tuberculosis isolates from mainland China.     

从结核病和肺癌患者外周血中检测结核分枝杆菌及其L型

目的 评价从外周血中检测结核分枝杆菌(MTB)及其L型(MTB-L)的临床应用价值.方法 用溶血离心滴片法IK抗酸染色(IK染色)及溶血离心培养法(培养法)检测结核病组(156例)、肺癌组(147例)和非结核病/非肺癌对照组(42例)外周血中的MTB及其L型,用TaqMan-PCR技术分别检测上述各组标本的单个核细胞、全血及血浆中的MTB DNA.结果 IK染色结核病组、肺癌组和非结核病/非肺癌对照组的MTB阳性率分别为1.3%(2/156)、0.7%(1/147)和0(0/42),培养法分别为0.6%(1/156)、0(0/147)和0(0/42).IK染色MTB-L阳性率分别为41.0%(64/156)、32.7%(48/147)和7.2%(3/42),培养法分别为25.6%(40/156)、39.5%(58/147)和0(0/42).结核病组、肺癌组的MTB与MTB-L检测结果 差异均有统计学意义(X2值分别为73.87、44.35、57.41、76.68,均P＜0.01).培养出98株L型菌中,人型81株,牛型17株,返祖为原菌型者44株.结核病组单个核细胞、全血TaqMan-PCR阳性率分别为77.6%(121/156)和68.6%(107/156),均高于血浆的阳性率的20.5%(32/156,X2值分别为112.96、82.18,均P＜0.05),肺癌组单个核细胞、全血阳性率分别为59.2%(87/147)和48.3%(52/147),均高于血浆阳性率的12.2%(18/147,X2值分别为70.53、21.68,P均＜0.05).两组各血液成分(除外血浆)的检测结果 与对照组比较差异均有统计学意义(X2值分别为37.50、48.30、44.37、11.31,P均＜0.01).结论 结核病、肺癌患者外周血中存在MTB-L播散;溶血离心培养法检测外周血中MTB-L简便易行;TaqMan-PCR技术检测单个核细胞和全血标本中MTB DNA的阳性率高于血浆的阳性率.     

Diagnostic approaches in patients with tuberculous pleural effusion

The conventional bacteriologic methods used for diagnosing pleural tuberculosis are less sensitive and time consuming. The objective of this study was to develop nonbacteriologic methods and to assess their potential utilities for the rapid diagnosis, especially in smear/culture-negative patients. One hundred forty patients with pleural effusion were investigated for tuberculous etiology by bacteriologic methods. Mycobacterium tuberculosis in the pleural fluid specimens was isolated in 11 patients. To establish a tuberculous etiology in the remaining 129 patients, we performed the following assays: a) estimation of tuberculosis-associated glycolipid antigen (TBGL) by a modified indirect enzyme-linked immunosorbent assay (ELISA), b) an immunocytochemical method for the demonstration of TBGL antigen in the Cytospin smears, and c) detection of mycobacterial DNA by polymerase chain reaction (PCR). Estimation of TBGL antigen by ELISA showed 100% specificity and overall 85.5% sensitivity. Immunocytochemistry could be applied only in those samples with adequate number of macrophages. PCR carried sensitivity and specificity of 87% and 93%, respectively. Estimation of TBGL antigen in pleural fluid specimens by ELISA has a definite role in establishing tuberculous etiology, particularly in those patients in whom bacteriologic methods did not demonstrate M. tuberculosis and also in those in whom a distinction between tuberculous and nontuberculous etiology was not possible based on the clinical and radiologic features of the thorax.     

Evaluation of direct method of drug susceptibility testing of Mycobacterium tuberculosis to rifampicin and isoniazid by nitrate reductase assay in a national reference laboratory

The aim of this study was to evaluate a simple, rapid, and inexpensive colorimetric nitrate reductase assay (NRA) for direct drug susceptibility testing (DST) of Mycobacterium tuberculosis against rifampicin (RIF) and isoniazid (INH). A total of 118 smear-positive specimens were processed from patients on antituberculosis treatment. A comparison was made between the direct NRA of DST with the direct proportion method and with the internationally accepted indirect 1% proportion method as the “gold standard”. The sensitivity and specificity of the direct NRA and indirect proportion method were 94% and 98%, and 100% and 98% for RIF and INH, respectively. Excellent agreement was found between the 2 tests with κ values of 0.92 and 0.98, and P value was less than 0.001 for RIF and INH. The results in most cases were available in 14 days (turnaround time). The direct NRA is a rapid, accurate, simple, and inexpensive method to determine multidrug resistance from sputum. Direct NRA may become an appropriate alternative method, especially for the resource poor settings.     

Rapid detection of Mycobacterium tuberculosis in sputum by Patho-TB kit in comparison with direct microscopy and culture

The usefulness of a new rapid diagnostic test (Patho-TB) using antibodies specific to mycobacterial antigens was evaluated for the rapid discrimination between pulmonary tuberculosis (TB) and non-TB pulmonary diseases on sputa. One hundred sputa collected from 79 active TB patients and from 21 patients with non-TB pulmonary diseases (asthma and chronic obstructive pulmonary disease) were enrolled into the study and tested for the presence of Mycobacterium tuberculosis by Ziehl–Neelsen smear, Patho-TB kit, and Löwenstein–Jensen culture. The sensitivity, specificity, positive predictive value, and negative predictive value of the Patho-TB test were 95%, 100%, 100%, and 84%, respectively. Patho-TB test is simple, quick, and easy to perform. Its sensitivity, specificity, and positive predictive value are satisfactory. Therefore, it could be used as a screening test in poorly equipped laboratories of TB endemic areas.     

耐多药结核分枝杆菌中embB基因突变与乙胺丁醇耐药的相关性研究

目的 研究耐多药结核分枝菌中embB基因突变与乙胺丁醇耐药的相关性. 方法 比例法检测84株耐多药结核分枝杆菌的乙胺丁醇(EMB)耐药性,基因测序检测embB基因的突变,2检验分析二者之间的相关性. 结果 84株耐多药结核分枝杆菌中有43株(51.2%)对EMB耐药,41株(48.8%)对EMB敏感,57株耐多药菌株(67.9%)的embB基因发生突变.在43株EMB耐药菌株中,embB基因突变的菌株为40株(93.0%),而41株EMB敏感菌株中,embB基因突变的菌株为17株(41.5%),embB基因在耐药菌株中的突变频率远高于敏感菌株(2=25.58,P=0.00).embB306是最常见的突变位点,其在耐药菌株的突变率也高于敏感菌株(2=12.37,P=0.00),embB基因和embB306位点检测EMB耐药的敏感度、特异度和准确性分别为93.0%和65.1%,58.5%和73.2%,76.2%和69.0%. 结论 EMB耐药的产生与embB基因和embB306突变有关,二者用于检测EMB耐药有一定的参考意义.     

结核分枝杆菌耐药的分子机制及分子生物学检测方法进展

结核分枝杆菌原发性和继发性耐药是世界范围问题,因此进行结核分支杆菌的耐药性机制和检测研究,对有效地防治结核病,尤其是指导临床用药上,具有极其重要的意义。结核分枝杆菌特异性突变位点是其耐药性的分子机制。