Evaluation of the diagnostic value of loop-mediated isothermal amplification assays targeting three different Mycobacterium tuberculosis genes.

BACKGROUND: The aim of the study was to evaluate a loop-mediated isothermal amplification (LAMP) assay for the ability to diagnose tuberculosis directly from clinical samples rapidly. METHODS: LAMP assays were performed using previously reported primer sets to amplify three specific Mycobacterium tuberculosis (MTB) gene targets, hspX, gyrB, and IS6110. Quantitated DNA from strain H37Rv were detected for assessment of analytical sensitivity; specificity was evaluated by testing eight species of non-tuberculosis Mycobacterium (NTM) and four unrelated bacterial species. Sputum samples from 68 pulmonary tuberculosis patients and a control group consisting of 45 lung cancer patients and 20 healthy controls were analyzed using LAMP assays, and then compared with smear, culture and quantitative real-time PCR (qRT-PCR) methods. RESULTS: All three LAMP assays showed 100% specificity for MTB when tested against NTM and other bacterial species. The gyrB-LAMP assay was able to detect 60 cfu/ml of H37Rv suspension within 1 h, similar to qRT-PCR, but 10 times more sensitive than the hspX-LAMP and IS6110-LAMP assays. In clinical samples, when qRT-PCR was used as the reference method, the sensitivity of the three LAMP assays targeting hspX, gyrB, and IS6110 genes was 94.6, 98.2 and 92.9%, respectively. CONCLUSIONS: LAMP is more sensitive than smear microscopy and close to qRT-PCR in sensitivity for the detection of MTB. LAMP has comparable specificity to qRT-PCR but was more rapid and convenient.

Performance of the Xpert® MTB/RIF assay in the rapid diagnosis of tracheobronchial tuberculosis using bronchial washing fluid.

OBJECTIVE: To assess the diagnostic value of the Xpert® MTB/RIF (GeneXpert) assay for tracheobronchial tuberculosis (TBTB) using bronchial washing fluid (BWF). METHODS: This retrospective study enrolled patients suspected of having TBTB and patients with non-TB pulmonary disease as controls. BWF were used to undertake acid-fast bacillus (AFB) smears, the GeneXpert assay and the LÓ§wenstein-Jensen (LJ) culture method. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were compared among BWF AFB smears, BWF GeneXpert and the BWF LJ culture method. RESULTS: A total of 130 patients with TBTB and 102 patients with non-TB pulmonary disease were enrolled in the study. Sputum AFB smears were positive in 62 of 130 patients (47.7%) with TBTB. Using the clinical diagnosis of TBTB as the gold standard, the sensitivity, specificity, PPV and NPV of the three methods using BWF were as follows: 93.1%, 99.0%, 99.2% and 91.8% for BWF GeneXpert; 73.1%, 100.0%, 100.0% and 74.5% for BWF LJ cultures; 53.8%, 99.0%, 98.6% and 62.7% for BWF AFB smears. The diagnostic yield of BWF GeneXpert was significantly higher compared with BWF cultures for type III and IV TBTB. CONCLUSION: The Xpert® MTB/RIF assay using BWF exhibited higher sensitivity than bacteriological diagnostic methods and was particularly useful for the early diagnosis of smear-negative TBTB.

[Evaluation of the reliability of thiophen-2-carboxylic acid hydrazine result for the identification of Mycobacterium bovis].

OBJECTIVE: To evaluate the reliability of thiophen-2-carboxylic acid hydrazine (TCH) test for identification of Mycobacterium bovis (M. bovis). METHODS: A total of 4069 clinically isolated strains were identified by P-Nitrobenzoic acid medium (500 mg/L) and TCH medium (5 mg/L). Mycobacterium tuberculosis (M. tuberculosis) complex strains susceptible to 5 mg/L TCH were further tested for susceptibility to 2 mg/L TCH. Spacer oligonucleotide typing (spoligotyping) and multi-loci PCR were also performed to identify TCH susceptible strains. RESULTS: Among the 4069 isolated strains there were 3929 strains belonging to M. tuberculosis complex (MTBC) of which 245 were susceptible to 5 mg/L TCH. Of these 245 strains, 20 were also susceptible to 2 mg/L TCH, while only 1 strain was identified to be M. bovis by both spoligotyping and multi-loci PCR. CONCLUSION: TCH susceptibility test (either 5 mg/L or 2 mg/L) is not a reliable method for identification of M. bovis.

Application of hyperbranched rolling circle amplification for direct detection of mycobacterium tuberculosis in clinical sputum specimens.

BACKGROUND: Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens. METHODS: A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb. RESULTS: The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72). CONCLUSIONS: Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

[Evaluation of the mycobacterium efficacy of linezolid in vitro].

OBJECTIVE: To evaluate the mycobacterium efficacy of linezolid to Mycobacterium tuberculosis bacilli and Non-tuberculous mycobacteria (NTM) in vitro, and to analyze the interaction between linezolid and other anti-TB drugs in vitro. METHODS: The minimum inhibition concentrations (MICs) of 121 Mycobacterium tuberculosis clinical isolates and 30 non tuberculosis Mycobacteria isolates and the corresponding standard strains to linezolid were tested by Microplate Alamar Blue assay (MABA). The interactions between linezolid and rifampicin, isoniazid, streptomycin, ethambutol, kanamycin, ofloxacin, and rifabutin were also tested in vitro by fractional inhibitory concentration index (FICI) method. RESULTS: 94.2% (114/121) of the Mycobacterium tuberculosis isolates were inhibited by linezolid at concentrations ≤ 1 mg/L. There was no statistical difference in the MIC values of sensitive strains, MDR strains, and drug resistant strains other than MDR (χ(2) = 0.481, P > 0.05). Only Mycobacterium kansasii was totally sensitive to linezolid among the 5 tested NTM strains. In vitro drug combination testing displayed overall non-association between linezolid and 7 other anti-TB drugs among 8 clinical isolates and H(37) Rv. CONCLUSIONS: Linezolid showed great mycobacterium efficacy to Mycobacterium tuberculosis clinical isolates in vitro, regardless of the strains' drug resistant parameters. This study also showed non-association of the interactions between linezolid and 7 other anti-TB drugs in vitro.

[An analysis on results of smear and culture of sputum from smear-positive tuberculosis patients].

OBJECTIVE: To study the positive rate of smear and culture in different sputa from smear-positive patients with tuberculosis, and to discuss the criteria of sputum selection for smear and culture. METHODS: The results of smear and culture of sputum samples from 3813 cases during tuberculosis drug-resistance baseline survey in 2007 were retrospectively analyzed. The positive rates of smear and culture of spot sputum, night sputum and morning sputum were compared, and the culture positive rate of sputum samples with 1-2 acid fast bacilli (AFB)/300 fields was summarized. The χ(2) test was used for comparison. RESULTS: The smear positive rate of spot sputum, night sputum, and morning sputum was 75.2% (2868/3813), 87.9% (3350/3813), and 89.4% (3408/3813) respectively. The percentage of cases which were positive for all the 3 sputum samples was 64.5% (2460/3813), while that for 2 samples and 1 sample was 23.4% (893/3813) and 12.1% (460/3813) respectively. The cumulative smear positive rate of spot sputum plus morning sputum reached 96.9% (3695/3813) of the rate combined from the 3 sputum samples. The culture positive rate of spot sputum, night sputum, and morning sputum was 97.8% (1654/1691), 96.7% (3006/3107) and 97.2% (2473/2543) respectively. For the smear positive cases, the positive rate of 2 sputum culture increased only 1.7% - 2.8% compared with that of 1 sputum culture. The culture positive rate of samples with a smear result of 1 - 2 AFB/300 fields was 90% (98/109). CONCLUSIONS: When spot sputum and morning sputum are collected for the diagnosis, the number needed for smear can be decreased from 3 to 2. For smear-positive cases, if sputum is selected for culture according to the smear result, the number needed for culture can be decreased from 2 to 1. A smear result of 1 - 2 AFB/300 fields is of considerable diagnostic value for tuberculosis.

Prevalence of tuberculosis drug resistance in 10 provinces of China.

BACKGROUND: The emergence of drug-resistant tuberculosis (TB) hampers TB control. Ten provinces in China performed drug resistance surveys among tuberculosis (TB) patients in 1996-2004 to assess levels of drug resistance. METHODS: Provincial drug resistance surveys included all isolates from newly diagnosed, smear-positive TB patients. Drug susceptibility testing (DST) against isoniazid, rifampicin, streptomycin and ethambutol was carried out in the provincial laboratories. For purposes of quality assurance, a random sample (11.6%) was re-tested by the national reference laboratory (NRL). RESULTS: Of 14,059 patients tested 11,052 (79%) were new TB cases. The weighted mean prevalence of multi-drug resistant tuberculosis (MDR-TB) among all cases was 9.3% (range 2.2%-10.4%); 5.4% (range 2.1% - 10.4%) among new cases and 25.6% (range 11.7%-36.9%) among previously treated cases. Adjusting the drug resistance proportions using the re-testing results did not change the estimated national mean prevalence significantly. However, in some individual provinces the estimated resistance proportions were greatly influenced, especially among re-treatment patients. CONCLUSION: MDR-TB levels varied greatly between provinces in China, but on average were high compared to the global estimated average of 4.8%. This study shows the importance of quality-assured laboratory performance. Programmatic management of drug-resistant TB, including high quality DST for patients at high risk of resistance and treatment with second-line drugs, should become the standard, especially in high MDR-TB settings.

[In vitro study on cross resistance of rifampin and rifapentine for Mycobacterium tuberculosis].

OBJECTIVE: To study the bactericidal effect of rifapentine and its cross-resistance with rifampin for Mycobacterium tuberculosis and to determine the critical concentration of rifapentine for laboratory drug susceptibility test and therefore to provide the laboratory data for using rifapentine in the treatment of tuberculosis, particularly rifampin resistant tuberculosis. METHODS: We detected the minimal inhibitory concentrations (MICs) of rifampin and rifapentine to H(37) Rv and isolated strains of rifampin susceptible and resistant Mycobacterium tuberculosis by using Middlebrook 7H9, Sauton and Lowenstein-Jensen media. RESULTS: The MICs of rifampin were > or = 0.32 microg/ml for 80% of the 19 rifampin susceptible strains on Middlebrook 7H9 and the MICs of rifapentine ranged from 0.02 microg/ml to 0.32 microg/ml for most of the strains (84%). The MICs of rifapentine were 2 - 4 times lower than those of rifampin to H(37) Rv and most clinical isolates. The rifapentine susceptible isolates were mostly separated from resistant strains at MICs 5-10 microg/ml. CONCLUSIONS: Our results demonstrate the cross resistance of rifampin and rifapentine and the stronger bactericidal potency of rifapentine than rifampin. Some rifampin resistant strains still show susceptibility to rifapentine, which suggests rifapentine may be effective in the treatment of rifampin resistant tuberculosis. Our results also determined a critical resistant concentration of rifapentine for routine drug susceptibility test.

[Characterization of the katG, inhA, ahpC, kasA, and oxyR gene mutations in isoniazid-resistant and susceptible strain of Mycobacterium tuberculosis by automated DNA sequencing].

OBJECTIVE: To study the characteristics of katG, inhA, ahpC, kasA, and oxyR gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. METHODS: A total of 101 isoniazid-resistant and 43 susceptible strains of Mycobacterium tuberculosis were analyzed by PCR and sequence analysis of their katG, inhA, ahpC, kasA, and oxyR genes. RESULTS: (1) Sequencing of katG from 101 INH-resistant strains showed point mutations, small deletions or insertions in 81 isolates (80.2%), but no complete deletions were identified. The mutations at 16 position were found for the first time. Point mutations at position 315 were found in the genomes of 38.6% (39/101) of isoniazid-resistant strains. Low level isoniazid resistant strains (1 microg/ml) had higher mutation frequency at 315-Ser than high level isoniazid resistant strains (10 microg/ml; chi(2) = 9.31, P < 0.05). Mutations at position 463 were detected in 58 (57.4%) isoniazid-resistant strains. Arg463leu was also present in 23 of 43 susceptible strains. (2) Mutations in inhA genes were identified in 5 isoniazid-resistant isolates (4.9%). None of the susceptible strains contained any mutation in inhA genes. (3) Only 3 isolates in the 101 (2.97%) isoniazid-resistant clinical isolates had mutations in ahpC genes. No mutations were identified in the ahpC genes in 43 isoniazid-susceptible isolates. (4) Mutations in kasA genes were present in 17 of 101 (16.8%) isoniazid-resistant isolates. However, G312S was also present in 3 of 43 susceptible strains. (5) None of the isoniazid-resistant strains and susceptible isolates contained oxyR gene mutation. (6) Taken together, 91 of 101 (90%) isoniazid-resistant strains had mutations in katG, inhA, ahpC, and kasA genes which were associated with drug resistance. CONCLUSION: These studies provide further evidence supporting the association between katG, inhA, ahpC, and kasA gene mutations and INH resistance in Mycobacterium tuberculosis, while other mechanisms of INH resistant may exist.

[Analysis and evaluation of phage amplified biologically assay, DNA sequencing analysis and single-strand conformational polymorphism for the drug susceptibility testing of Mycobacterium tuberculosis].

OBJECTIVE: To rapidly identify drug-resistant Mycobacterium tuberculosis using phenotypic and genotypic methods and to evaluate the clinical significance of rapid phenotypic susceptibility test by phage amplified biologically assay (PhaB). METHODS: PhaB, DNA sequencing and polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) were performed on the 91 rifampicin (RFP)-resistant strains (including 82 multi-drug resistant Mycobacterium tuberculosis strains), 42 RFP-susceptible strains, 75 ofloxacin (OFLX)-resistant strains and 40 OFLX-susceptible strains at the same time. RESULTS: The results obtained using PhaB assay, DNA sequencing and PCR-SSCP were compared with the absolute concentration method. For RFP susceptibility, the accordance, sensitivity and specificity of PhaB were 93%, 92% and 95% respectively; the accordance, sensitivity and specificity of DNA sequencing were 93%, 90% and 100% respectively; those of PCR-SSCP were 90%, 86% and 100% respectively. For OFLX susceptibility, the accordance, sensitivity and specificity of PhaB assay were 95%, 95% and 95%; those of DNA sequencing were 80%, 71% and 98% respectively; those of PCR-SSCP were 75%, 63% and 98% respectively. CONCLUSIONS: PhaB assay is a low-cost, rapid, and sensitive method and shows high accordance with absolute concentration technology. It can give drug susceptibility test results within 48 - 96 h, and is a promising technology in clinical laboratory.