Evaluation of rapid techniques for the detection of mycobacteria in sputum with scanty bacilli or clinically evident, smear negative cases of pulmonary and extra-pulmonary tuberculosis

The objective of the current study was to compare two rapid methods, the BBL Mycobacteria Growth Indicator Tube (MGIT TM) and Biotec FASTPlaque TB TM (FPTB) assays, with the conventional L?wenstein-Jensen (LJ) media assay to diagnose mycobacterial infections from paucibacillary clinical specimens. For evaluation of the clinical utility of the BBL MGIT TM and FPTB assays, respiratory tract specimens (n = 208), with scanty bacilli or clinically evident, smear negative cases and non-respiratory tract specimens (n = 119) were analyzed and the performance of each assay was compared with LJ media. MGIT and FPTB demonstrated a greater sensitivity (95.92% and 87.68%), specificity (94.59% and 98.78%), positive predictive value (94.91% and 99.16%) and negative predictive value (96.56% and 90.92%), respectively, compared to LJ culture for both respiratory tract and non-respiratory tract specimens. However, the FPTB assay was unable to detect nontuberculous mycobacteria and few Mycobacterium tuberculosis complex cases from paucibacillary clinical specimens. It is likely that the analytical sensitivity of FPTB is moderately low and may not be useful for the direct detection of tuberculosis in paucibacillary specimens. The current study concluded that MGIT was a dependable, highly efficient system for recovery of M. tuberculosis complexes and nontuberculous mycobacteria from both respiratory and non-respiratory tract specimens in combination with LJ media.     

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.     

Population Structure of Mixed Mycobacterium tuberculosis Infection Is Strain Genotype and Culture Medium Dependent

Background

Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection.

Aim

This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein–Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes.

Method

A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes.

Results

The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with “other genotype” strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype.

Conclusion

The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.     

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.     

Nontuberculous mycobacteria isolated from pulmonary specimens between 2004 and 2009: causative agent or not?

Nontuberculous mycobacteria were identified from 45891 samples of 19553 patients with a prediagnosis of pulmonary tuberculosis between November 2004 and January 2009. Among 10041 (21.9%) culture positive samples, 208 (2.1%) pulmonary samples recovered from 77 individual patients were differentiated as mycobacteria other than tuberculosis (MOTT). Proportion of mycobacteria evaluated as causative agent for clinical infection were found as 0.16% (n = 31), mostly M. avium complex, M. abscessus and M. kansasii. Additionally, M. fortuitum-peregrinum complex, M. simiae, M. szulgai / intermedium and M. scrofulaceum were found as causative agent in 2, 2, 2 and 1 patient, respectively. Identification of infections caused by environmental or opportunistic pathogen mycobacteria is required in rapid and accurate diagnosis, infection control and treatment planning of infections caused by M. tuberculosis complex and/or MOTT.     

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study

Background

Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.

Methods and Findings

The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001).

Conclusions

The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.     

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.     

Validation of a clinical-radiographic score to assess the probability of pulmonary tuberculosis in suspect patients with negative sputum smears

Background

Clinical suspects of pulmonary tuberculosis in which the sputum smears are negative for acid fast bacilli represent a diagnostic challenge in resource constrained settings. Our objective was to validate an existing clinical-radiographic score that assessed the probability of smear-negative pulmonary tuberculosis (SNPT) in high incidence settings in Peru.

Methodology/Principal Findings

We included in two referral hospitals in Lima patients with clinical suspicion of pulmonary tuberculosis and two or more negative sputum smears. Using a published but not externally validated score, patients were classified as having low, intermediate or high probability of pulmonary tuberculosis. The reference standard for the diagnosis of tuberculosis was a positive sputum culture in at least one of 2 liquid (MGIT or Middlebrook 7H9) and 1 solid (Ogawa) media. Prevalence of tuberculosis was calculated in each of the three probability groups.684 patients were included. 184 (27.8%) had a diagnosis of pulmonary tuberculosis. The score did not perform well in patients with a previous history of pulmonary tuberculosis. In patients without, the prevalence of tuberculosis was 5.1%, 31.7% and 72% in the low, intermediate and high probability group respectively. The area under de ROC curve was 0.76 (95% CI 0.72–0.80) and scores ≥6 had a positive LR of 10.9.

Conclusions/Significance

In smear negative suspects without previous history of tuberculosis, the clinical-radiographic score can be used as a tool to assess the probability of pulmonary tuberculosis and to guide the decision to initiate or defer treatment or to requesting additional tests.     

Detection of mycobacteria in clinical specimen using the mycobacteria growth indicator tube (MGIT) and the Lowenstein Jensen medium.

The recovery rates of mycobacteria strains isolated from 1200 clinical specimens using the mycobacteria growth indicator tube (MGIT) system and the conventional Lowenstein Jensen medium (LJ) were assessed. Of the 87 mycobacterial isolates recovered, 54 belonged to the M. tuberculosis complex (MTB) and 33 to the non-tuberculosis complex (NTM). MGIT recovered 78 (89.65%) mycobacteria isolates (51 MTB (94.44%) and 27 NTM (81.81%) and LJ recovered 70 (80.46%) mycobacteria isolates (49 MTB (90.74%) and 21 NTM (63.63%). Sixty one (70.1%) of the total mycobacteria isolates were recovered with both systems (46 (85.2%) MTB and 15 (45.5%) NTM). No significant difference was found between MGIT and LJ (p > 0.05) in both MTB and NTM recoveries. The average detection time for MTB was significantly shorter with MGIT than with LJ, in both the smear-positive specimens (8 vs 30 days: p < 0.0001) and smear-negative specimens (15 vs 30 days: p < 0.001). The average detection time of NTM was also shorter for MGIT (15 vs 30 days: p < 0.0001). However, the contamination rate was higher in MGIT (8.5%) than in LJ (3%). The results suggest that the use of MGIT contributes to a more rapid and effective diagnosis of mycobacterial infections particularly when combined with the classical LJ.     

Multi Drug and Other Forms of Drug Resistant Tuberculosis Are Uncommon among Treatment Na?ve Tuberculosis Patients in Tanzania

Background

Surveillance and effective management of drug resistance is important to sustaining tuberculosis (TB) control efforts. We aimed to determine resistance rates to first line anti tuberculosis drugs and to describe factors associated with the resistance to any of the first line anti tuberculosis drugs in Dar es Salaam Tanzania.

Materials

Newly diagnosed, TB patients with neither history of tuberculosis treatment nor isoniazid prophylaxis were included into the study. Sputum specimens were cultured on either mycobacteria growth indicator tube 960 (MGIT 960) or Lowenstein Jenstein (LJ) medium supplemented with either glycerol (GLJ) or pyruvate (PLJ). Drug susceptibility for isoniazid, rifampicin, streptomycin and ethambutol was determined by either Lowenstein–Jensen (LJ) medium or mycobacteria growth indicator tube 960 (MGIT 960).

Results

A total of 933 newly diagnosed TB patients, were included into the study. Multi drug resistance (MDR) tuberculosis was detected among 2 (0.2%) patients. Resistance to any of the four tested drugs was detected among 54 (5.8%) patients. Mono-resistance to isoniazid, rifampicin, streptomycin and ethambutol were 21(2.3%), 3 (0.3%), 13 (1.4%), 9 (1.0%) respectively.

Conclusion

Primary resistance to first line anti tuberculosis drugs is still low in this setting. Continued vigilance including periodic national surveillance of anti-tuberculosis resistance is recommended.     

Selective isolation of mycobacteria from soil: a statistical analysis approach

We compared four decontamination methods for the isolation of mycobacteria from soil specimens. Different media were used: L?wenstein-Jensen, Ogawa and various modified Ogawa media. Statistical analysis demonstrated that the best results (low contamination and high positivity rates) were obtained when the specimens were incubated in trypticase soy broth, treated with solutions containing malachite green and cycloheximide, then decontaminated with sodium hydroxide and inoculated onto Ogawa media. The lowest contamination rates were obtained with Ogawa medium containing 500 micrograms cycloheximide ml-1. The use of these techniques is proposed for the isolation of mycobacteria from heavily contaminated clinical specimens as well as from soil.     

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.     

应用GeneXpert MTB/RIF对肺外结核的快速检测及评估

目的 评估GeneXpert MTB/RIF检测肺外结核分枝杆菌的准确性,并与传统方法进行比较。 方法 选取2016年6月至2017年6月在本院就诊的144例疑似肺外结核病患者,对所有标本分别进行金胺“O”荧光染色镜检、液体培养及药敏试验、固体培养及比例法体外药敏试验和Xpert法检测。 结果 收集的144例疑似肺外结核标本中,确诊108例,以胸水、淋巴结活检和脓液感染较多,另36例阴性患者中,10例为非结核分枝杆菌感染。Xpert试验的敏感性为28.73%,特异性为96.00%,其阳性预测值和阴性预测值均高于其他3种检测方法。在阳性检出率方面,Xpert试验高于涂片镜检(χ2=17.39,P2=8.64,P2=2.56,P>0.05)。固体培养、液体培养和Xpert试验3种方法对结核分枝杆菌利福平耐药率检测差异无统计学意义(P>0.05),耐药率分别为8.33%、9.68%和11.11%,且Xpert试验方法检测出2株耐多药结核分枝杆菌,平均耗时2.5 h。 结论 GeneXpert MTB/RIF可以作为一种筛选及快速检测工具应用于肺外结核的诊断,同时可作为检测MDR TB的一种指标。     

Liquid vs Solid Culture Medium to Evaluate Proportion and Time to Change in Management of Suspects of Tuberculosis—A Pragmatic Randomized Trial in Secondary and Tertiary Health Care Units in Brazil

Background

The use of liquid medium (MGIT960) for tuberculosis (TB) diagnosis was recommended by WHO in 2007. However, there has been no evaluation of its effectiveness on clinically important outcomes.

Methods and Findings

A pragmatic trial was carried out in a tertiary hospital and a secondary health care unit in Rio de Janeiro City, Brazil. Participants were 16 years or older, suspected of having TB. They were excluded if only cerebral spinal fluid or blood specimens were available for analysis. MGIT960 technique was compared with the Lowenstein-Jensen (LJ) method for laboratory diagnosis of active TB. Primary outcome was the proportion of patients who had their initial medical management changed within 2 months after randomisation. Secondary outcomes were: mean time for changing the procedure, patient satisfaction with the overall treatment and adverse events. Data were analysed by intention-to-treat. Between April 2008 and September 2011, 693 patients were enrolled (348 to MGIT, 345 to LJ). Smear and culture results were positive for 10% and 15.7% of participants, respectively. Patients in the MGIT arm had their initial medical management changed more frequently than those in the LJ group (10.1% MGIT vs 3.8% LJ, RR 2.67 95% CI 1.44–.96, p = 0.002, NNT 16, 95% CI 10–39). Mean time for changing the initial procedure was greater in LJ group at both sites: 20.0 and 29.6 days in MGIT group and 52.2 and 64.3 in LJ group (MD 33.5, 95% CI 30.6–36.4, p = 0.0001). No other important differences were observed.

Conclusions

This study suggests that opting for the MGIT960 system for TB diagnosis provides a promising case management model for improving the quality of care and control of TB.

Trial Registration

Controlled-Trials.com ISRCTN79888843     

Impact of drug resistance on fitness of Mycobacterium tuberculosis strains of the W-Beijing genotype

Mycobacterium tuberculosis strains of the W-Beijing genotype became a common cause of tuberculosis during the past years and they are often associated with drug resistance. The biological factors facilitating the selection and wide dissemination of these strains are not known. To determine how acquisition of drug resistance affected growth of strains of the W-Beijing genotype, the growth of 55 M. tuberculosis isolates were studied using the BBL MGIT Mycobacteria Growth Indicator Tube and the BACTEC MGIT 960 System. Susceptible strains of non-Beijing genotypes were found to be the most fit strains. Drug-resistant strains of non-Beijing genotypes were more likely to grow slower than susceptible strains (P=0.001). Drug-resistant strains of the W-Beijing genotype had two tendencies of growth: some of them showed reduced growth compared to susceptible strains, while others did not show loss of fitness measured as growth.     

Fitness of Mycobacterium tuberculosis Strains of the W-Beijing and Non-W-Beijing Genotype

Background

Multidrug resistant tuberculosis (MDR-TB) is a major threat for global tuberculosis control. The W-Beijing Mycobacterium tuberculosis genotype has been associated with drug resistance. Elucidation of the mechanisms underlying this epidemiological finding may have an important role in the control of MDR-TB. The aim of this study was to evaluate the fitness of drug-susceptible and MDR M. tuberculosis strains of the W-Beijing genotype compared with that of Non-W-Beijing strains.

Methodology/Principal Findings

Fitness of M. tuberculosis strains was determined by evaluating the difference in the growth curves obtained in the MGIT960 automated system and assessing the competitive growth capacity between W-Beijing and non-W-Beijing strains. The W-Beijing MDR strains had a significant longer lag phase duration compared to the other strains but did not present a significant fitness cost. When grown in competition they had an advantage only in medium containing 0.1% Tween 80.

Conclusions/Significance

It was not possible to confirm a selective advantage of W-Beijing strains to grow, except for differences in their resistance to Tween 80. Further studies are needed to elucidate the putative advantage of W-Beijing strains compared to other genotypes.     

Clinical Utility of a Novel Molecular Assay in Various Combination Strategies with Existing Methods for Diagnosis of HIV-Related Tuberculosis in Uganda

Background

Low income, high-tuberculosis burden, countries are considering selective deployment of Xpert MTB/RIF assay (Xpert) due to high cost per test. We compared the diagnostic gain of the Xpert add-on strategy with Xpert replacement strategy for pulmonary tuberculosis diagnosis among HIV-infected adults to inform its implementation.

Methods

The first diagnostic sputum sample of 424 HIV-infected adults (67% with CD4 counts ≤200/mm³) suspected for tuberculosis was tested by direct Ziehl-Neelsen (DZN) and direct fluorescent microscopy (DFM); concentrated fluorescent microscopy (CFM); Lowenstein-Jensen (LJ) and Mycobacterial Growth Indicator Tube (MGIT) culture; and Xpert. Overall diagnostic yield and sensitivity were calculated using MGIT as reference comparator. The sensitivity of Xpert in an add-on strategy was calculated as the number of smear negative but Xpert positive participants among MGIT positive participants.

Results

A total of 123 (29.0%) participants were MGIT culture positive for Mycobacterium tuberculosis. The sensitivity (95% confidence interval) was 31.7% (23.6–40.7%) for DZN, 35.0% (26.5–44.0%) for DFM, 43.9% (34.9–53.1%) for CFM, 76.4% (67.9–83.6) for Xpert and 81.3% (73.2–87.7%) for LJ culture. Add-on strategy Xpert showed an incremental sensitivity of 44.7% (35.7–53.9%) when added to DZN, 42.3% (33.4–51.5%) to DFM and 35.0% (26.5–44.0%) to CFM. This translated to an overall sensitivity of 76.4%, 77.3% and 79.0% for add-on strategies based on DZN, DFM and CFM, respectively, compared to 76.4% for Xpert done independently. From replacement to add-on strategy, the number of Xpert cartridges needed was reduced by approximately 10%.

Conclusions

Among HIV-infected TB suspects, doing smear microscopy prior to Xpert assay in add-on fashion only identifies a few additional TB cases.     

Isolation and geographic distribution of Mycobacterium other than M. tuberculosis in British Columbia, 1972-81

It has been suggested that the incidence of infection with mycobacteria other than typical tubercle (MOTT) bacilli is increasing. Laboratory and epidemiologic information relating to MOTT infection in British Columbia between 1972 and 1981 was analysed. Patient records for 1960-81 were also analysed. Of the 313 661 laboratory specimens 13 474 yielded Mycobacterium tuberculosis isolates and 3172, MOTT isolates. Over the 10 years the number of M. tuberculosis isolates declined, whereas the absolute and relative numbers of MOTT isolates increased. Members of the highly drug-resistant MAIS complex (M. avium-intracellulare, M. scrofulaceum and M. simiae) accounted for 73.3% of the 1778 potentially pathogenic MOTT isolates. MAIS isolation rates varied geographically. Analysis of patient records revealed 217 MOTT infections, of which 152 (70%) were due to MAIS organisms. Further studies are needed to determine the source of MAIS organisms in order that the infection and the disease may be more clearly understood.     

Automatized PCR evaluation of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens

In this study, Mycobacterium tuberculosis complex isolates recovered from respiratory and nonrespiratory specimens with culture were evaluated using an automatized PCR method. Specimens with suspected tuberculous disease were decontaminated and concentrated using the standard N-acetyl-L-cysteine NaOH method and were inoculated onto glycerol-supplemented L?wenstein-Jensen media and BACTEC B12 vials. Forty-one specimens with typical colonies on solid media and 127 specimens identified as M. tuberculosis complex in a BACTEC system were selected as the study group. As the control group, 46 specimens without growth on either culture media were selected. The PCR results were positive in 33 (80.5%) and 87 (68.5%) samples that were culture-positive on solid and liquid media, respectively. All (100%) culture-negative specimens within the control group were also negative in the COBAS AMPLICOR Mycobacterium tuberculosis (MTB) PCR method. In conclusion, although it is a fast method for identifying M. tuberculosis complex isolates from clinical specimens, the COBAS AMPLICOR MTB PCR method is found to be less sensitive than culture techniques, we propose therefore that it should only be used in combination with culture results in the clinical diagnosis of tuberculosis.     

Detection of siderophores in growing cultures ofPseudomonas spp.

Summary The siderophores produced byPseudomonas fluorescens andP. chlororaphis were detected from the culture supernatants in MM9 and modified King's medium by the universal CAS assay at wavelengths 620–690 nm. The CAS assay was applied to detectPseudomonas siderophores directly in situ, during their production phase, in modified King's medium. Optimum results were detected with a final CAS concentration of 0.025 mM and an iron concentration of 1.25 M. The problems of the method are discussed with respect to the absorbance spectrum, the toxicity of the HDTMA detergent, the influence of the iron concentration and the complexity of media for siderophore production.