The results showed that the MGIT 960 system had a higher recovery rate of M. Bovis (122/129) than did the BACTEC 460 (102/129) and solid media system (96/129). The average time to detection was 15.8 days for the MGIT 960 system, 28.2 days for the BACTEC 460 system, and 43.4 days for solid media. The new BD BACTEC MGIT 960 System is the next generation in a proven line of mycobacteria testing instruments from BD Biosciences – the undisputed world leader in mycobacteriology. The BD BACTEC MGIT 960 System builds on the legacy of the BD BACTEC™ 9000MB System and the BD BACTEC™ 460TB System. BACTEC MGIT 960 System- Principle, Manual and Brochure The BACTEC MGIT 960 Mycobacterial Detection System is the world's first automated system for high-volume mycobacteria growth, detection and susceptibility testing from Becton Dickinson (BD). Scarparo C, Ricordi P, Ruggiero G, Piccoli P (2004) Evaluation of the fully automated BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide, streptomycin, isoniazid, rifampin, and ethambutol and comparison with the radiometric BACTEC 460TB method. J Clin Microbiol 42(3): 1109–1114 PMC free article.
The objective of the study was to compare the manual Mycobacteria Growth Indicator Tube (MGIT) method and the BACTEC MGIT 960 system to the BACTEC 460 method for susceptibility testing of Mycobacterium tuberculosis.The evaluation was based on testing of 36 M. Tuberculosis strains with various susceptibilities to isoniazid (INH), rifampin (RMP), ethambutol (EMB), and streptomycin (SM).
doi: 10.1128/JCM.01838-12
PMID: 23100351
This article has been cited by other articles in PMC.
Abstract
The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.
TEXT
Pyrazinamide (PZA) is a first-line drug currently being used for the treatment of both drug-susceptible and drug-resistant tuberculosis (TB) (). PZA (a nicotinamide analog) is a prodrug that requires conversion into its active moiety, pyrazinoic acid, by the mycobacterial enzyme pyrazinamidase (PZase) in order to be effective against Mycobacterium tuberculosis (, ). Loss of PZase activity is a common finding in PZA-resistant clinical isolates (, ), and mutations in pncA, the gene coding for PZase, are now regarded as the major mechanism of PZA resistance (–). In addition, the drug is active in an acid medium only, thus making drug susceptibility testing (DST) in the clinical laboratory more demanding (, ). Aiming to overcome the problem of frequent uninterpretable results because of poor growth, attempts have been made to set up a slightly acidified (pH 6), supplement-enriched medium able to support bacterial growth as well as to preserve PZA activity (, 12). In this context, the Bactec 460 radiometric system (Becton, Dickinson and Company, Sparks, MD), currently the “gold standard” method for PZA susceptibility (13), has been withdrawn from the market, and most clinical laboratories have already replaced or soon will replace it with the nonradiometric Bactec MGIT 960 (M960) system (Becton, Dickinson and Company, Sparks, MD). Although both methods utilize an acidified, 7H9-like broth and a modified proportion method with a critical concentration of 100 μg/ml, some recently published papers have reported unprecedented false resistance rates with the M960 system (–). Alternatively, PZA susceptibility may also be determined by testing cultured strains for PZase activity or by detecting pncA mutations. However, these methods also have their shortcomings: the PZase test lacks sensitivity (resistant strains may be PZase positive) (), and pncA mutations are highly diverse and widely scattered throughout the gene and regulatory region, thus limiting the chances to develop a simple and rapid commercial assay. Moreover, not all PZA-resistant M. tuberculosis isolates have mutations in the pncA gene (), and another gene (rpsA), coding for ribosomal protein S1, has recently been involved in a newly described mechanism of low-level PZA resistance (). The aim of the present study was to develop a method able to rule out major errors recently reported with the M960 system when testing the susceptibility of M. tuberculosis to PZA.
A total of 211 clinical isolates collected over a 2-year period from January 2010 to December 2011 were studied. Of these isolates, 201 were M. tuberculosis and 10 were Mycobacterium bovis. Microorganisms were identified to both the complex and species levels by standard procedures (19, ). Fully susceptible M. tuberculosis strain ATCC 27284 and PZA-resistant M. tuberculosis strain ATCC 35828 were used as reference strains.
PZA susceptibility testing using the MGIT 960 system was performed according to the manufacturer's instructions (Becton, Dickinson and Company, Sparks, MD) (21). Briefly, a positive MGIT tube obtained 1 to 2 days after the instrument had recorded a positive signal was used as the test inoculum. The test tube containing 0.1 ml of PZA solution in order to achieve the recommended critical concentration of 100 μg/ml was inoculated with 0.5 ml of the inoculum, while a drug-free control tube was inoculated with the same volume of a 1:10 dilution of the seed suspension. Tubes were placed in the MGIT 960 instrument and monitored until the control tube flagged positive. At that time, the PZA test tube was read as either resistant (≥100 growth units [GU]) or susceptible (<100 GU). PZA susceptibility testing with a reduced inoculum (RI) was performed as described above, except for the inoculum volume, which was reduced in both test and control tubes from 0.5 ml to 0.25 ml.
PZA testing using the radiometric Bactec 460 system was determined according to the instructions provided by the manufacturer (Becton, Dickinson and Company, Sparks, MD). Briefly, actively growing cultures exhibiting a growth index (GI) of greater than 300 were used as the inoculum source for the test. For each strain, two Bactec PZA medium vials were inoculated, one of which contained PZA (100 μg/ml) and polyoxyethylene stearate (POES) and the other of which was used as a control with POES only. The vials were incubated, and daily GIs were read on the Bactec 460 instrument until the GI of the control vial was ≥200. At that time, a strain was considered to be resistant if the GI of the PZA vial was >11% of the GI for the control vial, susceptible if <9%, and borderline in case of GI values ranging from 9 to 11% (22).
In order to evaluate the extent of PZA resistance, concentrations of 300 and 900 μg/ml were applied to those strains showing resistance to the critical concentration of 100 μg/ml. Moderate resistance was defined as a MIC of 300 μg/ml, while resistance as a MIC of ≥900 μg/ml was considered severe (12).
For amplification and sequencing of the pncA and rpsA genes, bacterial strains were grown on Löwenstein-Jensen slants at 37°C for 3 weeks. Colonies were suspended in 300 μl of distilled water, heated at 95°C for 20 min, incubated in an ultrasonic bath for 15 min, and centrifuged at 13,000 × g for 15 min. Five microliters of supernatants containing genomic DNA was used for PCR. A region of 932 bp containing the pncA gene was amplified in a C 1000 thermal cycler (Bio-Rad, Hercules, CA) using pncA_F3 (5′-AAGGCCGCGATGACACCTCT-3′) and pncA_R4 (5′-GTGTCGTAGAAGCGGCCGAT-3′) primers, with an initial denaturation step for 3 min at 95°C and 30 cycles as follows: 30 s at 95°C, 30 s at 55°C, and 1 min at 72°C, followed by a final 5-min step at 72°C (). A region of 1,530 bp containing the rpsA gene () was amplified using forward (5′-GTCCCTACGACCCAACCCTG-3′) and reverse (5′-GTCAGCCCGATGCGCAGCAT-3′) primers under the PCR conditions described above. Automated DNA sequencing was performed on a 3730 DNA analyzer and with 3730 Data Collection v.3 software (Life Technology, Paisley, United Kingdom) using the same primers and sequences aligned to wild-type H37Rv pncA with the ClustalW program.
After the standard M960 analysis, we observed PZA resistance in 55 out of 211 strains (26.1%). However, in a second M960 test which employed the RI procedure, 32 out of the 55 strains proved to be susceptible and resistance could be confirmed in 23 cases (10.9%), indicating a major error rate of 58.2% with the standard M960 test. Radiometric MICs of the presumptive true-positive strains showed that all of these except two exhibited severe resistance (>900 μg/ml), while a screening test applied to about 30% of discrepant isolates (10 out of 32), whose preliminary resistance was not confirmed with the RI assay, was negative (Table 1). Finally, follow-up testing of 14 true-resistant isolates (excluding 9 M. bovis isolates) showed the presence of pncA mutations in 12 of them (Table 2). The remaining two strains (showing a moderate degree of resistance) were further investigated for mutations in the rpsA gene, which did not reveal any nucleotide change upon sequencing analysis.
Table 1
Comparison of phenotypic and genotypic methods to detect pyrazinamide resistance in M. tuberculosis complex isolates
Mycobacterial species | No. of strains | No. of PZA-resistant isolates by: | No. of isolates with: | |||
---|---|---|---|---|---|---|
M960 | Bactec 460 | Radiometric MIC of >900 μg/ml | pncA mutation(s) | |||
Standard inoculum | Reduced inoculum | |||||
M. tuberculosis | ||||||
Fully susceptible | 169 | 33 | 5 | 5 | 3 | 3 |
Non-MDRa | 23 | 5 | 2 | 2 | 2 | 2 |
MDR | 9 | 7 | 6 | 6 | 6 | 6 |
M. bovis | 10 | 10 | 10 | 10 | 10 | NDb |
Total | 211 | 55 | 23 | 23 | 21 | 12c |
aMDR, multidrug resistant. One strain failed to grow in MGIT 960 (M960) PZA medium. It was shown to be susceptible when tested with Bactec 460.
cOf the 10 M. bovis strains, only one (recovered from a human source) was investigated for pncA mutations.
Table 2
Correlation between extent of phenotypic resistance and gene mutations in PZA-resistant M. tuberculosis and M. bovis isolates
Strain | Drug resistance patterna | Radiometric MIC (μg/ml) | pncA mutation | rpsA mutation | ||
---|---|---|---|---|---|---|
Site | Nucleotide change | Amino acid change | ||||
M. tuberculosis | ||||||
512151 | PZA | 300 | NAb | None (wild type) | NA | None (wild type) |
517108 | PZA | 900 | NA | None (wild type) | NA | None (wild type) |
551184 | PZA | >900 | 152 | A→G | His→Arg | NDc |
588707 | PZA | >900 | 464 | T insertion | Frameshift | ND |
589876 | PZA | >900 | 464 | T insertion | Frameshift | ND |
480717 | SM, INH, RMP, PZA | >900 | 61 | G insertion | Frameshift | ND |
373592 | SM, INH, RMP, EMB, PZA | >900 | 428 | C→G | Ala→Gly | ND |
530619 | SM, INH, RMP, EMB, PZA | >900 | 287 | A→T | Lys→Met | ND |
586492 | SM, INH, RMP, EMB, PZA | >900 | 291 | T deletion | Frameshift | ND |
589209 | SM, INH, RMP, EMB, PZA | >900 | 347 | T→C | Leu→Pro | ND |
589210 | SM, INH, RMP, PZA | >900 | 34 | G→A | Asp→Asn | ND |
589212 | INH, PZA | >900 | 289 | G→T | Gly→Cys | ND |
589216 | INH, PZA | >900 | 289 | G→T | Gly→Cys | ND |
M. bovis 521587 | PZA | >900 | 169 | C→G | His→Asp | ND |
aPZA, pyrazinamide; SM, streptomycin; INH, isoniazid; RMP, rifampin; EMB, ethambutol.
cND, not done.
Three phenotypic methods of PZA susceptibility testing were used in this study, with 211 isolates being tested by the standard M960 method, 55 by the RI assay, and 33 by the radiometric Bactec assay. When the test sensitivity and specificity for the above methods were calculated, assuming the radiometric system as the golden standard, the values were 100% and 100%, respectively, for the RI assay and 100% and 85.4%, respectively, for the standard M960 method. Cumulatively, the identification of PZA resistance within the pncA and rpsA genes exhibited a sensitivity and specificity of 92% and 100%, respectively. The overall accuracy of the RI assay for detecting PZA resistance was 100% (Fig. 1).
Diagnostic algorithm for detecting pyrazinamide resistance.
The nonradiometric M960 assay was shown to overreport PZA resistance, possibly because of several differences between the inoculum used in the M960 method and that used in the Bactec 460 assay. In fact, due to a different ratio of inoculum to medium, the bacillary concentration is more than 2.5 times greater in the M960 assay than that in the radiometric system (). Such a large amount of actively growing bacilli may significantly increase the pH of the culture medium, thereby inactivating the effect of PZA (). Moreover, there is an important variability in the concentration of the inoculum used in the Bactec M960 assay according to the day of test setup. For days 1 and 2 after the culture flags positive, there is no dilution of the MGIT seed vial, but for days 3 to 5, the inoculum becomes very heavy and must be diluted 1:5. However, even after dilution, large clumps are likely to crowd the inoculum, thus exceeding the rate of 106 CFU/ml required for a correct DST. In this context, a repeat testing using a 0.5-ml inoculum is discouraged because, besides poor reproducibility, it is likely to confirm false resistance results in as many as ≈50% of cases ().
Alternatively, a different approach may lie in reassessing the critical concentration for PZA susceptibility testing. Heifets has suggested that a resistance breakpoint of 300 μg/ml may be more appropriate than 100 μg/ml (, 12) with the Bactec radiometric method, and Zhang et al. considered a cutoff of 200 μg/ml closer to the theoretical MIC at pH 6.0 predicted from the Henderson-Hasselbach equation (), while Werngren et al., using the M960 system, proposed to classify clinical isolates as susceptible, intermediate, or resistant according to MIC values of <64 μg/ml, 128 μg/ml, and >128 μg/ml, respectively ().
To our knowledge, this is the first study in which major errors obtained testing PZA susceptibility with the M960 system have been solved by changing the inoculum rate. Our data show that a simple reduction from 0.5 ml to 0.25 ml allowed a clear separation between true- and false-resistant isolates, as demonstrated by full agreement with results obtained with the gold standard method. In addition, we routinely used disposable Pasteur pipettes to seed the inoculum (), which was dispensed regardless of any time-wasting procedure, such as leaving the seed MGIT to settle for 15 min and then taking the inoculum from the top of the seed tube instead of deeper down, close to the sediment. In case of severe resistance, our results were also in agreement with pncA mutation analysis, thus suggesting that molecular detection of PZA resistance is going to be the way forward for clinical laboratories as soon as rapid line probe assays are commercially available (, ).
In conclusion, due to the potential for major errors during PZA testing with the M960 assay, laboratories should consider retesting all PZA-resistant isolates to provide accurate and reliable susceptibility results. We suggest that any M. tuberculosis clinical isolate reported as PZA resistant by the standard M960 test should undergo a repeat DST using the RI of 0.25 ml. In case of confirmation, a pncA gene mutation analysis should be performed: otherwise, the isolate should be reported as susceptible.
Footnotes
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PMID: 10523555
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Abstract
The BACTEC MGIT 960 instrument is a fully automated system that exploits the fluorescence of an oxygen sensor to detect growth of mycobacteria in culture. Its performance was compared to those of the radiometric BACTEC 460 instrument and egg-based Lowenstein-Jensen medium. An identical volume of sample was inoculated in different media, and incubation was carried out for 6 weeks with the automatic systems and for 8 weeks on solid media. A total of 2,567 specimens obtained from 1,631 patients were cultured in parallel. Mycobacteria belonging to nine different taxa were isolated by at least one of the culture systems, with 75% of them being represented by Mycobacterium tuberculosis complex. The best yield was obtained with the BACTEC 460 system, with 201 isolates, in comparison with 190 isolates with the BACTEC MGIT 960 system and 168 isolates with Lowenstein-Jensen medium. A similar but not significant difference was obtained when the most-represented organisms, the M. tuberculosis complex, Mycobacterium xenopi, and the Mycobacterium avium complex, were analyzed separately and when combinations of a solid medium with the BACTEC MGIT 960 system and with the BACTEC 460 system were considered. The shortest times to detection were obtained with the BACTEC MGIT 960 system (13.3 days); 1.5 days earlier than that with the BACTEC 460 system (14.8 days) and 12 days earlier than that with Lowenstein-Jensen medium (25.6 days). The BACTEC MGIT 960 system had a contamination rate of 10.0%, intermediate between those of the radiometric system (3.7%) and the egg-based medium (17.0%). We conclude, therefore, that the BACTEC MGIT 960 system is a fully automated, nonradiometric instrument that is suitable for the detection of growth of tuberculous and other mycobacterial species and that is characterized by detection times that are even shorter than that of the “gold standard,” the BACTEC 460 system. The contamination rate was higher than that for the radiometric BACTEC 460 system and needs to be improved.
Although a variety of molecular biological methods have been shown to have the potential to provide direct detection of Mycobacterium tuberculosis complex from clinical specimens within a few hours (, ), culture still represents the cornerstone on which a definitive diagnosis of tuberculosis and other mycobacterioses relies. In recent years, the development of rapid, reliable methods for culture detection of acid-fast bacilli has been regarded as worthy of absolute priority (, ). Reasons for this renewed concern include the serious public health risk due to the reemergence of tuberculosis, the appearance of multidrug-resistant strains of M. tuberculosis, and the high incidence of Mycobacterium avium complex disease in patients with AIDS. Currently, mycobacterial culture can be performed with conventional solid media and by one of the available broth-based methods. Of these, the radiometric semiautomated BACTEC 460TB system (Becton Dickinson, Sparks, Md.), which was the first system to permit the significantly earlier detection of mycobacteria, is now widely accepted as the “gold standard” (4). It has several drawbacks, however: it involves the use of radioactive material, and reading of cultures is labor-intensive and is associated with a potential risk of cross-contamination. Furthermore the use of needles for inoculation of the vial involves the risk of stick injury. In recent years, several new nonradiometric technologies for growth and detection of acid-fast bacilli have been introduced; among these, the fluorimetric Mycobacteria Growth Indicator Tube (MGIT; Becton Dickinson) () and the MB-Redox system (Biotest, Dreieich, Germany) () are manual, while the ESP Culture System II (AccuMed, Chicago, Ill.) (, ), the MB/BacT system (Organon Teknica, Turnhout, Belgium) (), the BACTEC 9000 MB system (Becton Dickinson) (, ), as well as the BACTEC MGIT 960 system (Becton Dickinson) are fully automated, continuously monitoring, walk-away systems. Aha bls provider manual free download.
Both numbers reflect an average of user provided results as submitted to the. Higher numbers are better.You also might be interested in reviewing single core and multicore Geekbench 3 user submissions for devices with the iPhone3,2 Model Identifier, which may include.To dynamically compare 32-bit Geekbench 3 results from different iPod touch, iPhone and iPad models side-by-side, see Everyi.com's. The 'Discontinued Date' refers to the date a model either was replaced by a subsequent system or production otherwise ended.On October 4, 2011, Apple introduced this low-end 8 GB version of the and discontinued the earlier model. Iphone 4 model a1332 user manual download. Details:These Geekbench 3 benchmarks are in 32-bit mode and are for a single processor core and all processor cores, respectively. This final 8 GB revision was discontinued September 10, 2013 with the exception of the mainland China market, where it remained available for RMB 2,588 until September 9, 2014.Also see: All iPhone models introduced in.
The BACTEC MGIT 960 system is a noninvasive, nonradiometric system that uses the same technology used by manual MGIT and the BACTEC 9000 MB system. A ruthenium pentahydrate oxygen sensor embedded in silicon at the bottom of a tube containing 8 ml of modified Middlebrook 7H9 broth fluoresces following the oxygen reduction induced by aerobically metabolizing bacteria within the medium. A compact instrument incubates and tests, according to onboard algorithms, up to 960 culture tubes.
This paper summarizes the results of a multicenter clinical trial that compared the newly developed BACTEC MGIT 960 system, the radiometric BACTEC 460 system, and conventional solid medium for the recovery rates and time to detection of acid-fast bacilli from respiratory and extrapulmonary specimens.
MATERIALS AND METHODS
The investigation was carried on in three different Italian laboratories with 2,567 consecutive samples received with a request to determine the possible presence of mycobacteria. The specimens, most of which (94%) were smear negative, were obtained from 1,631 patients. Among the samples, 1,770 were respiratory (65% sputum samples, 22% bronchial aspirates, and 13% bronchial washings); among the nonpulmonary specimens, 380 were urine specimens and 137 were pleural fluid specimens, while 280 originated from various other body sites including stools, cerebrospinal fluid, ascitic fluid, pus, gastric juices, and biopsy specimens. While normally sterile body fluids (pleural fluid, pericardial fluid, cerebrospinal fluid, synovial fluid, and ascitic fluid) were concentrated by centrifugation only before being inoculated, respiratory specimens (sputum specimens, bronchial washings, and bronchoscopy specimens) and gastric fluid, urine, stool, pus, and tissue specimens were digested and decontaminated by the standard N-acetyl-l-cysteine–2% NaOH procedure (BBL MycoPrep; Becton Dickinson) (4). The supernatant was discarded, and the pellet was resuspended with sterile phosphate buffer to a final volume of 2 ml. The mixture was used both for preparation of a smear that was subsequently stained with auramine O and for inoculation of one BACTEC MGIT 960 tube (0.5 ml), one BACTEC 12B vial (0.5 ml), and two Lowenstein-Jensen slants (0.25 ml each).
Prior to inoculation BACTEC 12B and BACTEC MGIT 960 media were supplemented with the antibiotic mixture polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) and growth supplement (Becton Dickinson); vials to be inoculated with cerebrospinal fluid were not supplemented with PANTA, as suggested for the BACTEC 460 system. BACTEC MGIT 960 tubes were incubated at 37°C in the BACTEC MGIT 960 instrument, in which they were automatically monitored each hour for fluorescence development for 42 days or until a positive signal developed. The BACTEC 12B vials were incubated at 37°C and were monitored with the BACTEC 460 instrument (9) twice per week for the first 2 weeks and weekly thereafter for an additional 4 weeks or until the growth index (GI) was >10; bottles with GIs of >10 were monitored daily until the achievement of a GI of >100. Solid media were incubated at 37°C for 8 weeks and were inspected weekly or until mycobacterial colonies were seen.
BACTEC MGIT 960 tubes that had a positive signal with the instrument, BACTEC 12B vials with GIs of ≥100, and the growth of colonies on Lowenstein-Jensen medium were considered positive results only after confirmation of the presence of mycobacteria by means of an acid-fast smear. Positive cultures that failed to reveal acid-fast bacilli in the smear were screened, without concentration, for contaminants by Gram staining, and if positive, they were considered contaminated and eliminated, while, if negative, they were incubated again; acid-fast smears and Gram-stained smears were retested in BACTEC MGIT 960 tubes and signaled again as positive for a maximum two additional times. Thereafter, the cultures were considered false positive. Mycobacteria grown in culture were identified by using nucleic acid probes (AccuProbe; Gen-Probe, San Diego, Calif.) (11) and, when negative, were tested both by high-performance liquid chromatography (14) and by biochemical tests (4).
The rates of recovery were compared and analyzed by McNemar's chi-square test. The paired t test was used to compare times to detection.
RESULTS
A mycobacterium was isolated with at least one of the three culture systems from 236 samples corresponding to 109 patients; the majority of isolates were obtained from respiratory specimens (n = 184), while 53 mycobacteria were isolated from extrapulmonary sites, with the majority of them being obtained from gastric juice (11 isolates) and urine (9 isolates) specimens. Microscopy was positive for 129 specimens (54%).
Members of the M. tuberculosis complex were the most frequently isolated mycobacteria, followed by Mycobacterium xenopi and the M. avium complex; other nontuberculous mycobacteria were represented by nine Mycobacterium gordonae, six Mycobacterium chelonae, and three Mycobacterium malmoense isolates and one isolate each of Mycobacterium fortuitum, Mycobacterium kansasii, and Mycobacterium terrae. The identification to the species level of organisms of the M. tuberculosis and M. avium complexes allowed detection of six isolates of Mycobacterium bovis among the M. tuberculosis complex and, among the M. avium complex, isolates of M. avium, Mycobacterium intracellulare, and the MAI-X group () as well.
The comparison of rates of recovery by individual system is shown in Table Table1.1. The best yield was obtained with the BACTEC 460 system, but when compared with the BACTEC MGIT 960 system, it was not statistically significant; on the contrary, the two liquid media were significantly more sensitive than Lowenstein-Jensen medium both on the whole and among single species when the M. tuberculosis complex was considered separately.
TABLE 1
Recovery of mycobacteria by individual systems and combinations of systemsa
Mycobacterium or specimen | Total no. (%) recovered | |||||
---|---|---|---|---|---|---|
All media | BACTEC MGIT 960 system | BACTEC 460 system | Lowenstein-Jensen medium | BACTEC MGIT 960 system + Lowenstein-Jensen medium | BACTEC 460 system + Lowenstein-Jensen medium | |
All | 236 | 190 (80) | 201 (85) | 167 (71) | 212 (90) | 225 (95) |
M. tuberculosis complex | 169 | 149 (88) | 153 (92) | 124 (74) | 158 (94) | 160 (95) |
M. xenopi | 24 | 13 (54) | 11 (46) | 17 (71) | 21 (87) | 22 (92) |
M. avium complex | 22 | 21 (95) | 22 (100) | 16 (73) | 21 (95) | 22 (100) |
Other MOTTb | 21 | 7 (33) | 15 (71) | 10 (48) | 12 (57) | 21 (100) |
Smear positive | 228 | 122 (53) | 121 (53) | 103 (45) | 125 (55) | 127 (56) |
Smear negative | 108 | 68 (63) | 80 (74) | 64 (59) | 87 (80) | 98 (91) |
aMore frequently occurring taxa are considered separately, as were smear-positive and smear-negative specimens.
When the combinations of a liquid plus a solid medium were considered, the best performance was obtained with the BACTEC 460 system plus Lowenstein-Jensen medium (Table (Table1),1), which was more sensitive for the detection of both M. tuberculosis and other mycobacteria than the combination of the BACTEC MGIT 960 system and Lowenstein-Jensen medium (P = 0.04).
Fifty-two samples were found to be positive by only one method: 11 with the BACTEC 960 system, 24 with the BACTEC 460 system, and 17 with Lowenstein-Jensen medium (Table (Table2).2).
TABLE 2
Mycobacterium | No. of isolates detected | ||
---|---|---|---|
BACTEC MGIT 960 system | BACTEC 460 system | Lowenstein-Jensen medium | |
M. tuberculosis complex | 9a | 11a | 6 |
M. gordonae | 6 | 3 | |
M. xenopi | 2 | 3 | 6 |
M. avium | 1 | ||
M. chelonae | 1 | 1 | |
M. fortuitum | 1 | ||
M. kansasii | 1 | ||
M. terrae | 1 |
Twenty-three microscopically positive samples from which mycobacteria failed to grow on all three media were all obtained from previously culture-positive patients who had been treated.
The mean times to detection for paired samples grown by the three methods were 13.34 days (standard deviation [SD], 7.73 days; median, 12 days) with the BACTEC MGIT 960 system, 14.80 days (SD, 7.77 days; median, 14 days) with the BACTEC 460 system, and 25.67 days (SD, 11.55 days, median, 24 days) with Lowenstein-Jensen medium, with all differences being statistically significant. The separate times to detection of the most frequently isolated mycobacteria reported in Table Table33 emphasize the earlier detection with the BACTEC MGIT 960 system, which was moderate for M. tuberculosis complex (P = 0.03) but evident and highly significant for all nontuberculous mycobacteria. As expected, the times to recovery were shorter among smear-positive specimens (averages, 11.23 days with the BACTEC MGIT 960 system, 13.6 days with the BACTEC 460 system, and 22.91 days with Lowenstein-Jensen medium) than among smear-negative specimens (18.48, 19.00, and 32.38 days, respectively). For 20 samples, BACTEC MGIT 960 cultures gave a positive signal and the samples were incubated again, as no organism was detected in the broth by smear microscopy. The samples were found to be true positive about 5 or 6 days later and generally before the BACTEC 460 system gave a positive result.
TABLE 3
Mycobacterium and smear result | No. of strains | Average time to detection (days) | ||
---|---|---|---|---|
BACTEC MGIT 960 system | BACTEC 460 system | Lowenstein-Jensen medium | ||
Total | 137 | 13.34 | 14.80 | 25.67 |
M. tuberculosis complex | 113 | 14.25 | 14.88 | 25.08 |
Positive | 80 | 12.50 | 13.14 | 22.56 |
Negative | 33 | 19.58 | 19.12 | 31.18 |
M. xenopi | 4 | 21.75 | 29.75 | 55.25 |
Positive | 1 | 26 | 31 | 53 |
Negative | 3 | 20.33 | 29.33 | 56 |
M. avium complex | 16 | 5.94 | 8.56 | 23.31 |
Positive | 12 | 5.25 | 8 | 22.92 |
Negative | 4 | 8 | 4 | 24.50 |
Other MOTTa, all smear positive | 4 | 9 | 22.25 | 22.25 |
Cumulative detection times (Fig. (Fig.1)1) revealed both with the BACTEC MGIT 960 system and with the BACTEC 460 system a rate of positivity of over 80% within the 3rd week versus a rate of positivity of only 39% with Lowenstein-Jensen medium at the same time.
Cumulative percentages of mycobacteria detected weekly by individual methods. ▴, BACTEC MGIT 960 system; ●, BACTEC 460 system; ■, Lowenstein-Jensen medium.
The contamination rates were 3.70% with the BACTEC 460 system, 9.97% with the BACTEC MGIT 960 system, and 17.07% with Lowenstein-Jensen medium. Gram-positive cocci were the prevalent organisms responsible for contamination of BACTEC MGIT 960 tubes. For seven samples contaminants were also found in BACTEC MGIT 960 cultures positive for acid-fast bacilli. Fourteen samples had overgrowth in BACTEC MGIT 960 tubes but grew mycobacteria with at least one other culture medium. Contamination rates were practically the same at the three centers both for the BACTEC 460 system and for the BACTEC MGIT 960 system, while that for Lowenstein-Jensen medium was higher at one of the centers (22.4%). Four samples had false-positive results with the BACTEC MGIT 960 system; i.e., the tubes could not be tested to the end because of their repeated positive signals, even though they were negative for mycobacteria and contaminants by microscopy.
DISCUSSION
Rapid diagnosis of mycobacterial infections is critical; therefore, attempts to shorten the time needed for detection of such organisms deserve attention. The BACTEC MGIT 960 system is a fully automated, nonradiometric culture system which, due to continuous monitoring of O2 consumption, allows detection, without delay, of the mycobacteria growing within a liquid medium.
In our evaluation, the overall rates of recovery obtained with the BACTEC MGIT 960 and BACTEC 460 systems were clearly higher than those achieved with solid media, while in the comparison of the two liquid media, the rate of recovery of mycobacteria in the BACTEC MGIT 960 system was only slightly lower than that in the BACTEC 460 system. The use of one liquid medium and one solid medium is recommended by the Centers for Disease Control and Prevention (), and nowadays the use of such a combination is acknowledged worldwide. When the combination with the solid medium was considered, the difference between the BACTEC MGIT 960 system and the BACTEC 460 system was further reduced. Most of the differences among various methods were for mycobacteria other than M. tuberculosis.
In regard to turnaround times, the mean detection times were significantly shorter for methods that used a liquid medium than for Lowenstein-Jensen medium. The BACTEC MGIT 960 system detected positive samples an average of 1.5 days earlier than the BACTEC 460 system did. This is a statistically significant difference that was also confirmed when the most-represented mycobacterial species were considered singularly. Such times to detection are certainly affected by the different reading frequencies of various methods; a more frequent inspection of radiometric cultures and solid media is, however, incompatible with the laboratory routine and with the limited reading speed of the BACTEC 460 instrument, particularly in laboratories with high workloads. The continuous growth monitoring, which allows the real-time detection of positive cultures, far from being a bias factor, therefore represents an important feature of automatic systems like the BACTEC MGIT 960 system.
All potentially pathogenic mycobacteria encountered in this trial grew well in the BACTEC MGIT 960 system, including notoriously fastidious organisms like M. bovis and M. malmoense; the only species represented by more than one isolate which failed to grow in the BACTEC MGIT 960 system was M. gordonae, a well-known environmental contaminant.
The high rate of contamination of the BACTEC MGIT 960 system is probably due to the fact that this system uses a highly rich medium; the BACTEC 460 system, which relies on the high degree of sensitivity of radiometric detection, uses a less rich medium that is consequently less liable to overgrowth. Interestingly, we noticed that the contamination rate seemed to decrease during the study at all three centers that participated in the evaluation; such improvement suggests the need to set a period during which technicians can become accustomed to handling the screw-cap vials.
Of great interest is the fact that in 12 instances, what was counted here as contamination was due to the growth in the BACTEC MGIT 960 system of nocardiae and, in one further case, of a Rhodococcus sp.; these organisms, whose detection is clinically important because of their pathogenicity, particularly in immunocompromised patients, represented 5% of the contaminants.
Only one evaluation of the BACTEC MGIT 960 system has been published so far (). It reported recovery rates comparable to ours for the BACTEC MGIT 960 system and solid media and rates lower than ours for the BACTEC 460 system. The different ratio of the M. tuberculosis complex to the M. avium complex may well explain such a discrepancy as the M. tuberculosis complex growing better than the M. avium complex in the BACTEC 460 system. The M. tuberculosis complex represented more than 70% of our isolates but only 36% of the isolates in the other study. In both studies, but more evidently in ours, the BACTEC MGIT 960 system was characterized by the shortest times to detection. Although in both evaluations the contamination rate for the BACTEC MGIT 960 system was intermediate between those for the other methods, it appeared to be less favorable in our study than in another study.
Other automatic systems for the culture of mycobacteria have been introduced in recent times. Like with the ESP II system (), mycobacteria belonging to the M. avium complex appear to benefit more than M. tuberculosis with the BACTEC MGIT 960 system. On the contrary, M. xenopi, characterized by poor or absent growth both with the ESP II system () and the MB/BacT system (), grew easily and early in the BACTEC MGIT 960 system. On the other hand, the contamination rate for the BACTEC MGIT 960 system was the highest among those for automatic systems, thus emphasizing the higher risk of environmental contamination from the use of screw caps in comparison with that from the use of the rubber septum adopted by other systems. However, there are safety issues if needles are used for inoculation through a rubber septum.
The results reported here substantiate the fact that the BACTEC MGIT 960 system is a culture equivalent to the radiometric BACTEC 460 system. Recovery rates are very close to those of the radiometric method, while times to detection are even earlier. On the other hand, many good points characterize the system from the operative point of view: the radioactivity and the problems related to its use and disposal are not present, the full automation eliminates loading and unloading of tubes and minimizes the risk of bottle breakage, CO2 tanks are not required, the noninvasive monitoring of cultures eliminates the possibility of cross contamination, the use of screw caps on the tubes eliminate the need for use of needles and eliminates the risk of inadvertent needle pricks, the identification of samples by means of a bar code eliminates the risk of transcription errors, and maintenance is minimal. Furthermore, the space occupied by the BACTEC MGIT 960 instrument is very limited when one considers that it supports a heavy load (960 cultures corresponding to a daily capacity of 23 samples).
ACKNOWLEDGMENTS
We are grateful to Becton Dickinson for providing the instrumentation and reagents for evaluation.
We thank Uli Kunert and Salman Siddiqi for support.
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