MGIT System Research Articles

Comparative Study on Genotypic and Phenotypic Second-Line Drug Resistance Testing of Mycobacterium tuberculosis Complex Isolates

The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug- and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study of the current gold standard in the Netherlands, the Middlebrook 7H10 agar dilution method, the MGIT960 system, and the GenoType MTBDRsl genotypic method for rapid screening of aminoglycoside and fluoroquinolone resistance. We selected 28 clinical multidrug- and extensively drug-resistant M. tuberculosis complex strains and M. tuberculosis H37Rv. We included amikacin, capreomycin, moxifloxacin, prothionamide, clofazimine, linezolid, and rifabutin in the phenotypic test panels. For prothionamide and moxifloxacin, the various proposed breakpoint concentrations were tested by using the MGIT960 method. The MGIT960 method yielded results 10 days faster than the agar dilution method. For amikacin, capreomycin, linezolid, and rifabutin, results obtained by all methods were fully concordant. Applying a breakpoint of 0.5 microg/ml for moxifloxacin led to results concordant with those of both the agar dilution method and the genotypic method. For prothionamide, concordance was noted only at the lowest and highest MICs. The phenotypic methods yielded largely identical results, except for those for prothionamide. Our study supports the following breakpoints for the MGIT960 method: 1 microg/ml for amikacin, linezolid, and clofazimine, 0.5 microg/ml for moxifloxacin and rifabutin, and 2.5 microg/ml for capreomycin. No breakpoint was previously proposed for clofazimine. For prothionamide, a division into susceptible, intermediate, and resistant seems warranted, although the boundaries require additional study. The genotypic assay proved a reliable and rapid method for predicting aminoglycoside and fluoroquinolone resistance.

Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system

Mycobacterium tuberculosis reduces nitrate very strongly as compared to Mycobacterium bovis and M. bovis BCG. Nitrate reductase, in conjunction with niacin accumulation, constitutes one of the major biochemical tests used in clinical microbiology laboratories to differentiate M. tuberculosis from other members of the M. tuberculosis complex, as well as nontuberculous Mycobacteria. Determination of nitrate reductase activity is currently performed using cultures grown on solid media with a slow detection time and the need for large quantities of bacilli, as otherwise the test is not reliable. Hereby, we propose a nitrate reduction test coupled to Bactec MGIT960 system as a simple, rapid and economic method with a total gain of time of about 3 to 4 weeks over the conventional solid medium. In our study, almost all the M. tuberculosis and Mycobacterium canettii strains gave a strongly positive nitrate reductase result within 1 day of positive detection by the MGIT960 system. In contrast, M. bovis, M. bovis BCG and M. africanum strains remained negative even after 14 days of incubation. The possibility to detect nitrate reductase within 1 to 3 days of a positive culture using MGIT960 opens new perspectives with the possibility of confirming M. tuberculosis — starting directly from pathological specimens.

Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs

To evaluate the BACTEC(TM) MGIT(TM) 960/MGIT Para TB (MGIT) system for drug susceptibility testing of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogen implicated in some forms of Crohn's disease. MICs of 11 drugs for 10 MAP strains were determined using the MGIT system, the BACTEC(TM)460TB system (BACTEC) and conventional agar dilution methods. MICs determined by MGIT methods showed 80%-100% agreement (+/-1 log(2) dilution) with those determined by the BACTEC and agar dilution methods for ciprofloxacin, levofloxacin, azithromycin and clofazimine. The MGIT and BACTEC methods showed 70%, 80% and 90% agreement (+/-1 log(2) dilution) for MICs of ethambutol, rifabutin and rifampicin; agreement for all drugs increased to 100% at 2 log(2) dilution differences. For clarithromycin, the MGIT method had greater agreement with the agar dilution method (70% at the same dilution) than the BACTEC method (60% at +/-1 log(2) dilution); agreement increased to 100% at +/-2 log(2) dilutions in both cases. The MGIT and agar dilution methods agreed 60% and 100% for amikacin MICs at +/-1 log(2) dilution and +/-2 log(2) dilutions, respectively. By all methods MICs were higher than achievable serum concentrations for isoniazid and dapsone. There was 100% agreement between all three methods for azithromycin, clarithromycin and ciprofloxacin, and 80% agreement for rifampicin using published MIC thresholds available for M. avium complex strains. This study shows that the MGIT system can be used for rapid and reliable drug susceptibility testing of MAP.

Comparison of the conventional culture, the manual fluorescent MGIT system and the automated fluorescent MGIT 960 culture system for the detection of Mycobacterium avium ssp. avium in tissues of naturally infected hens

Different methods for the detection of Mycobacterium avium ssp. avium (MAA) in naturally infected hens were compared. They included the conventional culture method (solid Herrold's and Stonebrink media and liquid Sula medium) and newly developed liquid culture systems, the manual mycobacteria growth indicator tube (M-MGIT) and the fully automated BACTEC MGIT 960 system (A-MGIT). 152 tissues originating from 15 naturally infected hens have been processed. The overall detection rates (percentage of positive cultures from the number of positive cultures determined by all the methods together) were 60, 70 and 76 % for the conventional media, M-MGIT and A-MGIT systems, respectively, the mean time of mycobacteria detection being 32.6, 17.6 and 14.6 d, respectively. The lowest contamination rate (2.0 %) was found in A-MGIT compared with M-MGIT (4.6 %) and conventional media (10.4 %).

Comparison of Two Sputum Processing Methods for DetectingMycobacterium tuberculosisby Culture and PCR: Universal Sample Processing (USP) and NALC-NaOH Methods

Background: The universal sample processing (USP) method has recently been introduced as a simple technique that is applicable to smear microscopy, culture, and polymerase chain reaction (PCR) for the detection of Mycobaterium tuberculosis (MTB). The present study evaluated the utility of the USP meth- od for detecting MTB by culture and PCR, and the results were compared with that of the N-acetyl L-cysteine (NALC)-NaOH (6%) method. Methods: All sputum specimens were digested and decontaminated by both the USP and NALC-NaOH methods, and the processed samples were in- oculated for MTB culture and PCR. Culture was per- formed (252 samples) by using the MGIT system (Becton Dickinson Microbiology Systems, Sparks, Md, USA), and PCR test was conducted (281 sam- ples) by using Amplicor MTB kit (Roche Molecular Systems, Branchburg, N.J., USA). Results: MTB culture positive rates by NALC-NaOH and USP methods were 13.5% (34/252) and 11.9% (30/252), respectively (P>0.05). There were no sig- nificant differences between the two methods for de- tecting MTB by PCR: the MTB PCR sensitivities by USP and NALC-NaOH methods were 77.8% (49/63) and 82.5% (52/63), respectively, and the specificities were 95.9% (209/218) and 96.3% (210/218), re- spectively (P>0.05). Conclusion: There were no significant differences be- tween USP and NALC-NaOH methods of sample processing in enhancing the detection of MTB by culture or PCR. (Korean J Clin Microbiol 2009;12: 67-71)

Perianal involvement and inguinal adenitis as unusual presentation of tuberculosis

An 8-month old boy, a Rumanian gypsy immigrant to Spain, presented with a 2-month history of bilateral inguinal tumour and, at the beginning of this period, fever and vomiting. The mother was admitted for pulmonary tuberculosis with positive sputum smears in the infectious ward a week before the child. Examination revealed a well-nourished and afebrile child but in a bad hygienic condition. Both inguinal tumours were about 2x3 cm in size, painless and covered in normal cold skin. The left tumour fluctuated while the right one was hard and firmly adherent and, after 7 days, also became fluctuating (Fig. 1). The rest of the examination was unremarkable except for an erythematous perianal patch with a lineal ulcer (Fig. 2). The TST given was reactive (15 mm). The chest radiograph revealed a right hilar enlargement and computerized tomography (CT) demonstrated mediastinal adenopathy (10 mm) in the right hilum, an inflammatory infiltrate in the right anterior segment. The pleura was not affected. Acid-fast stains of the gastric juices were negative. The blood coagulation analysis was altered (prothrombin time 11.1 s, fibrinogen 575 mg/dl and APTT 57.2 s), and showed an unspecific decrease of intrinsic system factors. Serological tests for HIV, toxoplasma, and CMV were negative. In addition to tuberculous lesions, the differential diagnosis in this patient included lymphoma, and sexual abuse lesions. A skin biopsy from the perianal lesion revealed a chronic granulomatous inflammation. Acid fast stains of skin biopsy and FNA of the inguinal nodes were negative. Due to the unclear preliminary results, a lymphadenectomy was undertaken [5] and sample yielded also negative for stain. With the MGIT system (BD), a mycobacterial culture of the cutaneous and surgical specimens became positive. DNA probes (GenProbe) demonstrated the organism to be within the Mycobacterium tuberculosis complex. The isolate was shown to be susceptible to antituberculous agents. A regimen of rifampicin and isonizazid for 6 months, plus pyrazinamide for the first 2 months was initiated. Streptomycin was also included until susceptibility tests were available because of the risk of resistance owing to the country of origin [2]. This antibiotic was then discontinued. Eur J Pediatr (2007) 166:967–968 DOI 10.1007/s00431-006-0324-8

Stepwise Decrease in Moxifloxacin Susceptibility amongst Clinical Isolates of Multidrug-ResistantMycobacterium tuberculosis:Correlation with Ofloxacin Susceptibility

To study the effectiveness of moxifloxacin (MOX) against multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB), in vitro drug susceptibility tests on MOX and ofloxacin (OFX) using the MGIT system against 132 nonduplicate MDR MTB isolates (108 OFX-sensitive and 24 OFX-resistant) were performed. Eleven OFX-resistant non-MDR MTB isolates were also included. All strains that were susceptible to OFX were shown to be also susceptible to MOX. For OFX-resistant isolates, regardless of their MDR status, a 4- to 8-fold decrease in MIC was observed for MOX when compared to OFX. On the basis of similarities in mode of action and pharmacokinetic data between MOX and OFX, a breakpoint MIC of 2 mg/L was suggested for MOX in the MGIT system. All but two OFX-resistant isolates possessed MIC of MOX of 2 mg/L or lower and were considered to be susceptible to MOX; however, all OFX-resistant strains studied had mutations involved in resistance to quinolones. DNA sequence analysis for the gyrase A mutation, which was an indicator for OFX resistance, correlated with MOX susceptibility changes. Significant decreases in MOX susceptibility amongst MDR-MTB strains were observed that correlated with OFX susceptibility. The Asp94Gly mutation appeared to be associated with a higher level of MICs to both OFX and MOX. These findings on the stepwise decline of in vitro susceptibility to MOX suggest that clinical usage of MOX should be accompanied by careful monitoring of susceptibility to this important anti-MDR MTB drug.

The Mycolic Acids Analysis with Hplc Technique in Drug Susceptibility Testing of Mycobacterium Tuberculosis Strains

The aim of this study was to evaluate the utility of the quantitative analysis of mycolic acids by HPLC technique in drug susceptibility testing of the M. tuberculosis isolates to the first-line antituberculous drugs: isoniazid and rifampicin. Drug susceptibility of the 30 clinical M.tbc isolates was examined by the mycolic acids analysis with HPLC technique and results were compared to the proportion method on solid L-J medium and liquid medium in MGIT system. In HPLC method drug susceptibility of M. tuberculosis strains was described by TAMA index defined as the ratio of thetotal area under mycolic acids peaks (TAMA) from cultures with drug to the TAMA of control. At critical concentrations of drugs, TAMA indexes of resistant strains were >0.5, and TAMA indexes of susceptible strains were <0.05. The average error of the TAMA analysis was ±9.5% The quantitative analysis of mycolic acids by HPLC gives results compatible with standard proportion method and is a reliable method for determination of drug susceptibility of M. tuberculosis.

Rapid, automated, nonradiometric susceptibility testing of Mycobacterium tuberculosis complex to four first-line antituberculous drugs used in standard short-course chemotherapy

The increasing prevalence of drug-resistant tuberculosis necessitates rapid and accurate susceptibility testing. The nonradiometric BACTEC Mycobacteria Growth Indicator Tube 960 (MGIT) system for susceptibility testing was evaluated on 222 clinical Mycobacterium tuberculosis complex isolates for isoniazid, rifampin, and ethambutol. Fifty-seven of the isolates were tested for pyrazinamide. Results were compared to those of radiometric BACTEC 460 system and discrepancies were resolved by the agar proportion method. We found an overall agreement of 99.0% for isoniazid, 99.5% for rifampin, 98.2% for ethambutol, and 100% for pyrazinamide. After resolution of discrepancies, MGIT yielded no false susceptibility for rifampin and isoniazid. Although turnaround times were comparable, MGIT provides an advantage as inoculation can be done on any weekday as the growth is monitored automatically. The automated MGIT system is a rapid and reliable alternative for susceptibility testing of M. tuberculosis complex to first-line drugs.

Evaluation of the fully automated Bactec MGIT 960 system for the susceptibility testing of Mycobacterium tuberculosis to first-line drugs: a multicenter study

The accuracy of the Bactec MGIT 960 system for susceptibility testing of 177 clinical isolates of Mycobacterium tuberculosis to first line drugs (isoniazid, rifampicin, ethambutol and streptomycin) was compared with the agar reference method. The sensitivity, the ability to detect resistance, of the MGIT system was 100%, while the specificity, the ability to detect susceptibility, ranged from 98.6% to 100% for all drugs tested.

Evaluation of the BACTEC MGIT 960 in comparison with BACTEC 460 TB for detection and recovery of mycobacteria from clinical specimens

The BACTEC MGIT system (M960), a fully automated, non radiometric instrument, designed for rapid detection of acid fast bacilli (AFB) from clinical specimens, was compared with the radiometric BACTEC 460 system (B460) and Löwenstein-Jensen (L-J) solid medium. A total of 1,093 respiratory and extrapulmonary specimens were decontaminated by the NALC-NaOH standard method, and randomly inoculated into the media. A total of 122 mycobacteria were recovered, including 47 Mycobacterium tuberculosis complex (MTB) isolates and 75 nontuberculous mycobacteria (NTM) isolates. Overall recovery rates were 59% for M960 system ( p < 0.0001), 58.2% for L-J. medium ( p < 0.0001) and 82% for Bactec 460 system, whereas rates for MTB alone were 91.5, 76.6 ( p = 0.007), and 95.7%, respectively. The combination of M960 or B460 systems with L-J medium showed the same recovery rates for MTB strains (97.9%), whereas NTM rates were 68% ( p < 0.0001) and 93.3%, respectively. Mean time to detection of smear-positive MTB, smear-negative MTB, and NTM were 12.2, 13.4, and 23.3 days, respectively, with the M960, 11.7, 21.3, and 24.8 days with the B460 and 20.4, 28.7, and 28.4 days with L-J medium. The M960 system showed a contamination rate of 9.8%, while B460 and L-J medium showed contamination rates of 4.3 and 3.8% respectively. In conclusion, the M960 system appeared to be accurate and rapid for the recovery of MTB, but reduced recovery of NTM and a high number of contaminated cultures deserve further study in order to assess if this system can represent a valuable alternative to the radiometric system.

Evaluation of a new method for rapid drug susceptibility testing of Mycobacterium avium complex isolates by using the mycobacteria growth indicator tube.

The reliability of the Mycobacteria Growth Indicator Tube (MGIT [BBL]) for rapid drug susceptibility testing of Mycobacterium avium complex (MAC) isolates was evaluated. MICs of amikacin, clarithromycin, clofazimine, ethambutol, and rifabutin were determined by the MGIT system for 16 MAC strains. The results were compared with those obtained by the BACTEC broth macrodilution method. The turnaround times were 6 to 8 days (median, 7 days) for the MGIT and 5 to 7 days (median, 6 days) for the BACTEC system. Agreements with BACTEC system-determined MICs, within +/-1 log2 dilution, were 100, 100, 88, 63, and 44% for amikacin, clofazimine, rifabutin, clarithromycin, and ethambutol, respectively. Within +/-2 log2 dilutions, agreement with BACTEC system-determined MICs increased to 100% for all the tested drugs. In addition, if MGIT-determined MICs were evaluated according to the thresholds adopted for the interpretation of BACTEC system-determined ones, ethambutol was the only drug for which susceptible strains were frequently misclassified as resistant. It is concluded that the MGIT system is a promising, nonradiometric alternative to the BACTEC method for rapid susceptibility testing of MAC isolates; however, additional studies are required to confirm our results and to determine the optimal criteria for the interpretation of ethambutol MICs.