Direct identification of Mycobacterium avium complex and Mycobacterium gordonae from MB/BacT bottles using AccuProbe

We evaluated the ability of the AccuProbe (Gen-Probe, San Diego, Calif.) to detect Mycobacterium gordonae and Mycobacterium avium complex directly in liquid medium flagged positive by the MB/BacT (Organon Teknika Corp., Durham, N.C.). Seventy-one bottles from clinical specimens containing M. gordonae and 34 containing M. avium, confirmed by culture, were tested by direct AccuProbe assay for both organisms after additional incubation for > or = 48 h and centrifugation at 4,500 x g for 15 min. Relative light unit (RLU) values were analyzed using the manufacturer's recommended cutoff of 30,000 RLU and a lower cutoff of 10,000 RLU. Using the 30,000 RLU cutoff, 55 of 71 (77.5%) specimens containing M. gordonae yielded positive results, whereas 28 of 34 (82.3%) M. avium complex specimens were correctly identified by direct probe. No specimens shown by culture to contain either M. gordonae or M. avium complex tested positive with the probe for the opposite organism (100% specificity). When the cutoff was lowered to 10,000 RLU, 67 of 71 M. gordonae (94.4%) and 32 of 34 M. avium complex (94.1%) specimens were correctly identified. This difference was significant for M. gordonae (P = 0.004) but not for M. avium complex (P = 0.26) compared to detection using the recommended RLU cutoff. Specificity was 100% for specimens containing M. gordonae that were tested with the M. avium complex probe using the 10,000 RLU cutoff, whereas specificity for specimens containing M. avium complex tested with the M. gordonae probe was 97%. Using a lower RLU cutoff for determining a positive result using the M. gordonae or M. avium complex probes when testing instrument-positive MB/BacT bottles directly will improve sensitivity without substantially compromising specificity.     

Use of nucleic acid probes for identification of Mycobacterium tuberculosis directly from MB/BacT bottles

The feasibility of using nucleic acid probes directly from positive MB/BacT broth to identify mycobacteria was determined in this study. A total number of 2,727 specimens were cultured into the MB/BacT (Organon Teknika) automated system and on conventional Loweinstein-Jensen (LJ) slants. The Gen-Probe AccuProbe culture identification tests (DNA probes) were used on samples from bottles which were identified as positive for mycobacteria by MB/BacT. Samples of positive MB/BacT broth (0.1 ml) were used directly in the broth culture method for the DNA probes as published by Gen-Probe. Centrifugation of the contents of the bottle was not done prior to probe testing. The number of mycobacteria detected by MB/BacT and LJ was 253 (221 isolates of M. tuberculosis and 32 isolates of mycobacteria other than M. tuberculosis [MOTT]). A total of 96.4% (213 of 221) of the bottles growing M. tuberculosis produced a positive direct DNA probe result for M. tuberculosis complex. One hundred percent (16 of 16) of the bottles growing M. gordonae produced a positive direct DNA probe result for M. gordonae. A total of 3.6% (8 of 221) of the bottles growing M. tuberculosis did not yield a positive direct DNA probe result for M. tuberculosis complex. The testing of subcultures made onto solid media from the positive bottles by AccuProbe identified six of these eight M. tuberculosis isolates. Two (0.9%) M. tuberculosis isolates gave a negative result for the M. tuberculosis probe test applied on the MB/BacT broth and its subculture. The rest of the positive MB/BacT bottles growing MOTT (16 of 32) were negative for M. gordonae, M. avium, M. intracellulare, and M. kansasii probes. The sensitivity and specificity of AccuProbe for the identification of M. tuberculosis and M. gordonae directly from MB/BacT broth were 96.4 and 100% for M. tuberculosis and 100 and 100% for M. gordonae, respectively. The direct testing of positive MB/BacT broth by AccuProbe, without prior centrifugation, allows for the accurate and rapid identification of M. tuberculosis and M. gordonae.     

Comparison of the MB/BacT and BACTEC 460 TB systems for recovery of mycobacteria from various clinical specimens

A total of 1,830 specimens (75.7% respiratory and 24.3% nonrespiratory) were cultured in parallel with the MB/BacT and BACTEC 460 TB systems and on Lowenstein-Jensen (LJ) medium. Mycobacteria were identified from 173 (6.5%) specimens. The most common species recovered were Mycobacterium tuberculosis complex (65. 9%), Mycobacterium avium complex (22.5%), and Mycobacterium chelonae (9.2%). The recovery rates by individual systems were 96.5, 99.4, and 95.9% for MB/BacT, BACTEC 460 TB, and LJ medium, respectively, for all mycobacteria; the recovery rates were 99.1, 100, and 98.2%, respectively, for M. tuberculosis complex alone. The difference among the recovery rates for all mycobacteria and those for individual species was not significant. The BACTEC 460 TB system detected M. tuberculosis isolates more rapidly than the MB/BacT system (8 versus 11.8 days for smear-positive specimens [P < 0.01] and 18 versus 21 days for smear-negative specimens [P < 0.05]), whereas the MB/BacT system more rapidly detected the nontuberculous mycobacteria (17.1 versus 12.7 days [P < 0.01]). These results indicate that the nonradiometric MB/BacT system is a rapid, sensitive, and efficient method for the recovery of M. tuberculosis and nontuberculous mycobacteria from both pulmonary and extrapulmonary clinical specimens.     

Controlled evaluation of BacT/alert standard anaerobic and FAN anaerobic blood culture bottles for the detection of bacteremia and fungemia.

FAN medium was formulated to improve microbial recovery, particularly for fastidious microorganisms and for microorganisms causing sepsis in patients receiving antimicrobial therapy. In a controlled clinical evaluation performed at four university-affiliated hospitals, FAN anaerobic bottles were compared with standard anaerobic bottles for yield, speed of detection of microbial growth, and detection of septic episodes. A total of 10,431 blood culture sets were received; both anaerobic bottles of 7,694 blood culture sets were adequately filled with blood. Altogether, 925 isolates were recovered: 557 that were the cause of sepsis, 99 that were indeterminate as the cause of sepsis, and 269 contaminants. More Staphylococcus aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Escherichia coli (P < 0.02), and all microorganisms combined (P < 0.005) were recovered from FAN bottles; more nonfermentative gram-negative bacilli (P < 0.05), Torulopsis glabrata (P < 0.001), and other yeasts (P < 0.01) were recovered from standard bottles. Growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Enterococcus faecalis (P < 0.025), streptococci other than Streptococcus pneumoniae (P < 0.01), and all microorganisms combined (P < 0.001) was detected earlier in standard bottles; growth of more isolates of E. coli (P < 0.05) and anaerobic bacteria (P < 0.01) was detected earlier in FAN bottles. The mean times to detection were 14.2 and 16.1 h for standard and FAN bottles, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative evaluation of Vitek 2 identification and susceptibility testing of Gram-negative rods directly and isolated from BacT/ALERT-positive blood culture bottles

The performance of Vitek 2 was evaluated for the identification and susceptibility testing of Gram-negative bacilli directly from positive blood cultures bottles. Direct inoculation of the positive blood cultures with the Vitek cards ID-GN and AST-NO58 was compared with the standard inoculation method based on the sub-culture of the positive blood culture to agar. A total of 142 blood cultures were included in the study; of those, 119 were from patients’ clinical samples, while 23 were artificially prepared with strains showing different mechanisms of resistance. A total of 136 (95.8%) strains were correctly identified to the species level, only 2 (1.4%) were mis-identified and 4 (2.8%) were not identified. Susceptibility results were available for all isolates tested against 17 antibiotics, thus, resulting in a total of 2,414 isolate/anti-microbial combinations. The error rate was 2.8% (67/2,414) overall; 0.6% (14/2,414) very major errors, 0.1% (3/2,414) major errors and 2.1% (50/2,414) minor errors. The direct method detected 88.5% (22/25) of the strains producing extended-spectrum beta-lactamases (ESBLs). The susceptibility agreement among the added strains with ESBL, AMPc hyperproduction, resistance to ceftazidime, carbapenems and cefepime was very high. Direct identification and susceptibility testing gave rapid and reliable results, reducing by 24 h the turnaround time of the microbiology laboratory.     

Evaluation of the BACTEC MGIT 960 and the MB/BacT systems for recovery of mycobacteria from clinical specimens and for species identification by DNA AccuProbe

A total of 120 mycobacterial isolates were recovered from 1,068 clinical specimens. Of these, 82.5% were in MGIT 960, 83.3% were in MB/BacT, 80% were in BACTEC 460, and 70% were on L?wenstein-Jensen medium. Mean times to detection of Mycobacterium tuberculosis (n = 96) were significantly shorter with MGIT 960 (12.6 days, P = 0.003) and BACTEC 460 (11.8 days, P < 0.001) than with MB/BacT (15.9 days). Although, MGIT 960 showed the lowest rate of recovery of M. kansasii genotype I (64.3%), the earliest growth was detected with this system (8.9 days). Low and similar rates of contamination were obtained with MGIT 960 (3.3%) and MB/BacT (3%). The AccuProbe test for identification showed excellent sensitivities with MGIT 960 (96. 8%) and MB/BacT (100%) cultures. In addition to being nonradiometric, both MGIT 960 and MB/BacT are accurate, rapid, and labor-saving detection systems which could replace the radiometric method.     

Rapid identification of Staphylococcus aureus from BacT/ALERT blood culture bottles by direct Gram stain characteristics

The rapid identification of Staphylococcus aureus from positive blood cultures provides important clinical and therapeutic information. Using criteria based on direct Gram stain characteristics, an experienced microscopist was able to distinguish S aureus from other staphylococci isolated from BacT/ALERT blood culture bottles with an overall sensitivity of 89% and specificity of 98%. Furthermore, this method was readily taught to a clinical microbiologist who had not previously used the method first hand. Laboratories using the BacT/ALERT blood culture system should become familiar with these criteria so that S aureus bacteraemia can be identified rapidly.     

Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and genotype Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with Mycobacterial strains

The performance of two DNA line probe assays, a new version of INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium) and the GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), were evaluated for identification of mycobacterial species isolated from liquid cultures. Both tests are based on a PCR technique and designed for simultaneous identification of different mycobacterial species by reverse hybridization and line probe technology. The INNO-LiPA Mycobacteria v2 targeting the 16S-23S rRNA gene spacer region was developed for the simultaneous identification of 16 different mycobacterial species. The GenoType Mycobacterium, which targets the 23S rRNA gene, allows simultaneous identification of 13 mycobacterial species. Both tests were evaluated on 110 mycobacterial strains belonging to 22 different mycobacterial species (20 reference strains, 83 clinical strains, and 4 Mycobacterium kansasii strains isolated from tap water) that were previously inoculated into MB/BacT bottles. The sensitivity of both methods, defined as the number of positive results obtained with the Mycobacterium genus probe together with an interpretable result on the number of samples tested was 110 of 110 (100%) for INNO-LiPA and 102 of 110 (92.7%) for GenoType. For samples with interpretable results, INNO-LiPA was able to correctly identify 109 of 110 samples (99.1%), whereas the GenoType correctly identified 100 of 102 samples (98.0%). Both tests were easy to perform, rapid, and reliable when applied to mycobacterial identification directly from MB/BacT bottles.     

Evaluation of the MB/BacT System for recovery of mycobacteria from clinical specimens in comparison to Lowenstein–Jensen medium

Objective   To evaluate the performance of the MB/BacT system (Organon Teknika) in comparison to Lowenstein–Jensen (LJ) solid medium for recovery of mycobacteria from clinical specimens.
Methods   Two thousand three hundred and ten specimens (1626 respiratory, 593 urine, 60 body fluids, five tissue and 26 others) were inoculated in MB/BacT (0.5 mL) and on two LJ slants (0.25 mL each). N -acetyl- l -cysteine-NaOH (final concentration 2%) was used for decontamination.
Results   Two hundred and fifty-one (10.8%) mycobacterial isolates [190 Mycobacterium tuberculosis complex (MTBC) and 61 non-tuberculous mycobacteria (NTM)] were detected. Of these 251 isolates, 234 (93.2%; 181 MTBC and 53 NTM) were detected in MB/BacT and 169 (67.3%; 154 MTBC and 15 NTM) on LJ. The mean (median) times to detection of MTBC by MB/BacT and LJ were 13.8 (13) and 22.1 (20) days, respectively, while overall contamination rates were 7.7% and 8.1%, respectively.
Conclusions   Sensitivity and time to detection were significantly better with MB/BacT than with solid LJ medium.     

Direct identification of Staphylococcus aureus from positive blood culture bottles

Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targeting Staphylococcus aureus 16S rRNA is a novel method for direct identification of S. aureus from positive blood culture bottles. The test (S. aureus PNA FISH) is performed on smears made directly from positive blood culture bottles with gram-positive cocci in clusters (GPCC) and provides results within 2.5 h. A blinded comparison of S. aureus PNA FISH with standard identification methods was performed in collaboration with eight clinical microbiology laboratories. A total of 564 routine blood culture bottles positive for GPCC recovered from both aerobic and anaerobic media from three different manufacturers (ESP, BACTEC, and BacT/Alert) were included in the study. The sensitivity and specificity of S. aureus PNA FISH were 100% (57 of 57) and 99.2% (116 of 117), respectively, with 174 GPCC-positive ESP blood culture bottles, 98.5% (67 of 68) and 98.5% (129 of 131), respectively, with 200 GPCC-positive BACTEC blood culture bottles, and 100% (74 of 74) and 99.1% (115 of 116), respectively, with 190 GPCC-positive BacT/Alert blood culture bottles. It is concluded that S. aureus PNA FISH performs well with commonly used continuously monitoring blood culture systems.     

Rapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles by using the LightCycler system

One hundred BacT/ALERT blood culture bottles growing gram-positive cocci in clusters were cultured and studied by LightCycler PCR for the sa442 and mecA genes. PCR was 100% sensitive and specific for detecting Staphylococcus aureus and methicillin resistance in S. aureus but was less accurate for methicillin resistance in coagulase-negative staphylococci.     

Evaluation of the MB/BacT system and comparison to the BACTEC 460 system and solid media for isolation of mycobacteria from clinical specimens.

The MB/BacT automated system is designed for the isolation of mycobacteria from clinical specimens. It utilizes a colorimetric sensor and reflected light to continuously monitor the CO2 concentration in the culture medium. We compared its performance to that of the BACTEC 12B media for the radiometric BACTEC 460 instrument and that of solid culture media. Respiratory specimens and urine samples were decontaminated with 4% NaOH. The vials of the two instruments were inoculated with 500 microl of sample and two solid egg-based media at 200 microl each. All vials were incubated at 37 degrees C for 6 weeks. A total of 1,078 specimens (633 respiratory specimens, 78 cerebrospinal fluid specimens, 177 other body fluid specimens, 87 urine specimens, and 103 other types of specimens) were cultured in parallel. Mycobacteria could be identified from 73 (6.8%) specimens: 67 M. tuberculosis, 3 M. kansasii, 1 M. xenopi, 1 M. terrae, and 1 mixed M. avium with M. scrofulaceum. Of these, 63 (86.3%) specimens were positive with the MB/BacT system, 67 (91.8%) were positive with the BACTEC 460 instrument, and 58 (79.5%) were positive with the two egg-based media. MB/BacT cultures were positive on average after 17.5 (+/-6.4) days, BACTEC cultures with a growth index of >20 (mean, 200) were positive after 14.3 (+/-8.2) days, and egg-based media were positive after 24.2 (+/-7.5) days. Microorganisms other than mycobacteria contaminated 46 (4.3%) MB/BacT cultures and 31 (2.9%) BACTEC cultures, which had to be discarded. The MB/BacT system is a well-automated system for the detection of M. tuberculosis in clinical specimens without using radioactive reagents. Further trials are required to determine whether it is suitable for the culture of nontuberculous mycobacteria.     

Evaluation of BacT/Alert 3D liquid culture system for recovery of mycobacteria from clinical specimens using sodium dodecyl (lauryl) sulfate-NaOH decontamination

A total of 52 mycobacterial isolates were recovered from 1,197 clinical specimens decontaminated by a sodium dodecyl (lauryl) sulfate (SDS)-NaOH protocol. Of these, 94% were recovered with the BacT/Alert 3D system (Organon Teknika, Durham, N.C.) and 79% were recovered on L?wenstein-Jensen (LJ) medium. Mean times to detection of organisms of the Mycobacterium tuberculosis complex (n = 47) were 22.8 days with LJ medium and 16.2 days with the system. The BacT/Alert 3D system is a rapid and efficient detection system which can be used with an SDS-NaOH decontamination procedure.     

Controlled evaluation of 5 versus 10 milliliters of blood cultured in aerobic BacT/Alert blood culture bottles.

Bottles developed for use in the BacT/Alert automated blood culture system (Organon Teknika Corp., Durham, N.C.) can accept up to 10 ml of blood without falling below a 1:5 ratio of blood to broth. We compared the yield and speed of detection of microorganisms in 13,128 adequately filled, paired, aerobic bottles inoculated with 5 versus 10 ml of blood at three university hospitals. A total of 798 microorganisms causing sepsis grew in one or both bottles. The overall recovery of microorganisms from 10-ml samples exceeded that from 5-ml samples (P < 0.001); the increased yield attributed to the additional 5 ml in the samples was 7.2%. The increased yield from 10-ml inocula was most marked for Escherichia coli (P < 0.01) and other members of the family Enterobacteriaceae (P < 0.001). Ten-milliliter samples did not yield more gram-positive bacteria, nonfermentative gram-negative rods, or yeasts. When both bottles were positive, the bottles inoculated with 10 ml of blood showed growth sooner (P < 0.001). Earlier detection with 10-ml inocula was especially notable for coagulase-negative staphylococci (P < 0.001), streptococci (P < 0.001), E. coli (P < 0.025), and other members of the family Enterobacteriaceae (P < 0.025). We conclude that an increase in the volume of blood inoculated into BacT/Alert aerobic blood culture bottles from 5 to 10 ml will increase the overall yield and the speed of detection of clinically important blood pathogens.     

Comparative recovery of microorganisms from BacT/ALERT plastic and glass FA and FN blood culture bottles

bioMerieux, Inc., has recently introduced plastic bottles to replace glass bottles for use in the BacT/ALERT blood culture system. We compared the performance of the plastic to the glass bottles in a large clinical evaluation. Two blood cultures were collected from each patient, one using glass FA (aerobic) and FN (anaerobic) bottles and one using plastic FA and FN bottles. Of the 4,040 sets of four bottles collected, 3,110 contained the recommended 8 to 12 ml of blood, yielding 524 microorganisms with 359 judged to be clinically significant. Of the 359 significant organisms, 255 were recovered in either one or two bottles from both pairs of bottles in a set while 56 organisms were recovered only from the glass bottles and 48 were recovered only from the plastic bottles (P, not significant [NS]). Of the 286 significant organisms recovered only in the FA bottles (glass and plastic), 180 were recovered in both bottles, 57 in the plastic bottles only, and 49 in the glass bottles only (P, NS). Of the 303 significant organisms recovered in the FN bottles only (glass and plastic), 212 were recovered in both bottles, 46 in the plastic bottles only, and 45 in the glass bottles only (P, NS). For individual organisms, the only significant difference in recovery was obtained for Escherichia coli, with more isolates recovered in the FN plastic than in the FN glass bottles (P = 0.02). These data suggest that recovery of microorganisms with plastic FA/FN bottles is at least equal to that with glass FA/FN bottles while offering greater safety for users.     

Evaluation of BacT/Alert 3D automated unit for detection of nontuberculous mycobacteria requiring incubation at 30 degrees C for optimal growth

The reliability of the BacT/Alert 3D unit for automated detection of nontuberculous mycobacteria (NTM) that grow optimally at 30 °C was assessed. This system reliably maintained a temperature of 30 °C and detected 50% of the clinical NTM strains (5 Mycobacterium marinum and 3 Mycobacterium gordonae strains) faster than 37 °C culture.     

Direct identification of bacteria from charcoal-containing blood culture bottles using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Several protocols for direct matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) from positive blood cultures are currently used to speed up the diagnostic process of bacteraemia. Identification rates are high and results are accurate for the BACTEC? system and for charcoal-free bottles. Only a few studies have evaluated protocols for charcoal-containing BacT/ALERT bottles reaching substantially lower identification rates. We established a new protocol for sample preparation from aerobic and anaerobic positive charcoal-containing BacT/ALERT blood culture bottles and measured the protein profiles (n?=?167). Then, we integrated this protocol in the routine workflow of our laboratory (n?=?212). During the establishment of our protocol, 74.3?% of bacteria were correctly identified to the species level, in 23.4?%, no result and in 2.4?%, a false identification were obtained. Reliable criteria for correct species identification were a score value?≥1.400 and a best match on rank 1-3 of the same species. Identification rates during routine workflow were 77.8?% for correct identification, 20.8?% for not identified samples and 1.4?% for discordant identification. In conclusion, our results indicate that MALDI-TOF MS is possible, even from charcoal-containing blood cultures. Reliable criteria for correct species identification are a score value?≥1.400 and a best match on rank 1-3 of a single species.     

Controlled clinical evaluation of BACTEC Plus Aerobic/F and BacT/Alert Aerobic FAN bottles for detection of bloodstream infections.

A total of 7,190 blood culture sets were obtained from adult patients with a suspected bloodstream infection. A 20-ml sample of blood was distributed equally between the aerobic FAN bottle which was monitored in the BacT/Alert system and a Plus Aerobic/F bottle which was monitored in the BACTEC 9240 system. A total of 988 positive cultures were obtained from 483 patients; however, only 453 positive cultures from 173 patients met the criteria for volume ( > or = ml per bottle) and clinical significance on the basis of concurrent case review required for data analysis. There were 25 and 68 false positives from the FAN and Plus Aerobic/F bottles, respectively. There were no statistically significant differences between systems in the number of positive cultures or septic episodes by species; however, the total number of Enterobacteriaceae and Pseudomonas aeruginosa isolates combined was significantly greater in the FAN bottle (P = 0.04). Detection times did not differ significantly between systems for positive cultures; however, episodes of Staphylococcus aureus bacteremia were detected significantly more rapidly from the FAN bottle (P = 0.005). There was no significant difference between systems in the detection of bloodstream infections in patients receiving antibiotics at the time of blood culture.     

Direct comparison of the BACTEC 9240 and BacT/ALERT 3D automated blood culture systems for candida growth detection

A direct comparison of two automated blood culture systems was conducted to compare their ability to detect Candida growth. The systems evaluated were the BACTEC 9240 (Bactec) and BacT/ALERT 3D (BacT). The aerobic, anaerobic, and mycology media for each system were evaluated: Bactec Plus Aerobic/F, Plus Anaerobic/F, and Myco/F Lytic bottles, respectively, and BacT FA, SN, and MB bottles, respectively. Each blood culture bottle was inoculated with fresh blood from healthy donors. Fifty isolates of Candida spp. were used. The six different blood culture bottles were each inoculated with 1000 yeasts per bottle and then incubated in the corresponding automated system. The BacT detected growth of 90% (135 of 150) of Candida pathogens, while Bactec detected 66% (100 of 150). Growth was detected in all BacT and Bactec mycology bottles, all BacT aerobic bottles, and by terminal subculture of all bottles. Sixty-five of 300 (22%) bottles had no growth detected; 50 from the Bactec (5 aerobic and 45 anaerobic) and 15 from the BacT (all anaerobic). Terminal subculture of "negative" bottles demonstrated viable yeast growth from all 65 bottles, representing 65 false-negatives. The mean time to growth detection in the BacT system was 25.62 h while the Bactec was 27.30 h (P < 0.01). Both automated blood culture systems detected all episodes of simulated candidemia when specialized mycology media were used. However, when only standard aerobic and anaerobic media were used, the BacT performed better than the Bactec in overall growth detection, time to growth detection, and number of false-negatives.