Evaluation of CrystalTM Rapid Stool/Enteric ID System for Identification of Aerobic Gram-negative Bacilli

Objective: The evaluation of the new miniaturized Crystal^TM Rapid Stool/Enteric System (Becton-Dickinson, USA) for identification of aerobic gram-negative bacilli.
Methods: a total of 154 clinical organisms ( Enterobacteriaceae: 120 strains; oxidase-positive fermenters: 13 strains; non-fermenters: 21 strains) were tested. Results were compared with those obtained with the PASCO^R system (Difco, USA) and divergent identifications were evaluated by standard biochemical tests.
Results: without additional testing, correct identification was obtained for 146 strains ( Enterobacteriaceae: 95%; oxidase-positive fermenters 87%; non-fermenters 100%). For adequate identification of Yersinia enterocolitica strains, however, panels had to be incubated for 5 instead of 3 hours.
Conclusions: the Crystal^TM Rapid Stool/Enteric system offers a safe, accurate and rapid method for the identification of frequent isolates of the family Enterobacteriaceae and bacterial stool pathogens.     

Evaluation of Biolog system for identification of some gram-negative bacteria of clinical importance.

The Biolog system (Biolog, Inc., Hayward, Calif.) was evaluated for the identification of 55 gram-negative taxa (789 strains) likely to be encountered commonly in clinical laboratories. The Biolog system performed best with oxidase-positive fermenters and biochemically active nonfermenters and had the most problems with unreactive nonfermenters. It gave significantly better results when the MicroPlates were read manually rather than when they were read by the automated reader. Plates read manually gave the following performances: oxidase-positive fermenters, five taxa, 64 strains, 92% correct, 3% not identified, and 5% incorrect; biochemically active nonfermenters, eight taxa, 122 strains, 88% correct, 6% not identified, and 6% incorrect; members of the family Enterobacteriaceae, 35 taxa, 511 strains, 77% correct, 8% not identified, and 15% incorrect; unreactive nonfermenters, seven taxa, 92 strains, 38% correct, 24% not identified, and 38% incorrect. We found the system easy to use, but while for 39 of 55 of the taxa an identification rate of > 70% was achieved, problems were encountered, particularly with identification of capsulated strains of some Enterobacter and Klebsiella taxa, as well as the least biochemically active Moraxella and Neisseria strains.     

Evaluation of a new identification system, Crystal Enteric/Non-Fermenter, for gram-negative bacilli.

A total of 505 fermentative and 201 nonfermentative gram-negative bacilli, identified by conventional methods, were tested by the Crystal Enteric/Non-Fermenter ID kit and by the API 20E or API 20NE identification system. The overall correct results for fermenters were 92.9% by the Crystal kit and 89.1% by the API 20E system. The false identifications (genus and species incorrect) accounted for 3.1 and 7.1% for the Crystal and API systems, respectively. For nonfermenters, figures for correct identifications by the two systems were comparable (Crystal, 75.9%; API 20NE, 75.3%) while the API 20NE system gave twice as many incorrect results (13.8%) as Crystal (6.3%); however, Crystal failed to precisely identify several species included in a "miscellaneous" group. The Crystal Enteric/Non-Fermenter system is an easy-to-use kit which compares favorably with other commercial systems.     

Evaluation of PASCO MIC-ID system for identifying gram-negative bacilli.

A production model of the semi-automated PASCO MIC-ID system (PASCO Laboratories, Wheat Ridge, Colo.) was evaluated with 122 groups or species of gram-negative bacilli, which included typical (499 cultures) and atypical (37 cultures) strains of fermenters and nonfermenters. The PASCO system identified 90.9% of 536 cultures accurately; these included 90.8% of 152 nonfermenters, 93.8% of 308 enteric fermenters, and 78.9% of 76 oxidase-positive fermenters. These results were obtained with the aid of serologic tests and a few additional biochemical tests, when recommended by the PASCO system. Of the 14 misidentified nonfermenters, 3 were Pseudomonas paucimobilis, 3 were Weeksella virosa (Centers for Disease Control group IIf), 2 were Xanthomonas (Pseudomonas) maltophilia, and 6 were randomly distributed among the other groups and species tested. The 19 enteric fermenters that were misidentified were randomly distributed among the groups and species tested. Of the 16 misidentified oxidase-positive fermenters, 4 were Pasteurella ureae, and 12 were randomly distributed among the other groups and species. The system identified the most commonly encountered organisms at a rate of 95% or better. The PASCO system is easy to inoculate and read. A slightly improved data base should remedy most of the identification problems.     

Evaluation of the updated QUANTUM II system for the identification of gram-negative bacilli.

The QUANTUM II system (Abbott Diagnostics, Irving, Tex.) was evaluated with 65 species of gram-negative bacilli from various culture collections at the Centers for Disease Control. The QUANTUM II system accurately identified 92.5% of 335 isolates tested, as follows: 92.6% of 258 members of the family Enterobacteriaceae, 92.7% of 55 nonfermenters, and 91% of 22 oxidase-positive fermenters. These results were obtained by using the additional biochemical and serologic tests recommended by the manufacturers of the QUANTUM II system. The 25 misidentified cultures generally belonged to newly recognized genera, atypical strains, or slower-growing strains of more widely known genera. The system identified the most commonly encountered organisms at an accuracy of greater than or equal to 95%. The system is efficient, accurate, and rapid.     

autoSCAN-4 system for identification of gram-negative bacilli.

A production model of the autoSCAN-4 system (American MicroScan, Inc., Mahwah, N.J.) was tested with not more than 11 strains each of 73 groups or species of gram-negative bacilli from various Centers for Disease Control culture collections. The strains included typical and atypical strains of enteric fermenters, nonenteric fermenters, and nonfermenters. The autoSCAN-4 system identified 95.3% of all 405 cultures accurately: 95.4% of 307 members of the family Enterobacteriaceae, 96.6% of 29 nonenteric fermenters, and 94.2% of 69 nonfermenters. Manual readings of the same trays provided essentially the same results, with a maximum change of only +1.6% identification accuracy of members of the Enterobacteriaceae. These data were obtained by all required additional tests, including serology and computer consultation when indicated. Only 19 of the cultures tested were misidentified. These were distributed randomly throughout the various groups and species except that Edwardsiella tarda was usually missed because of poor H2S reactions in the test medium. Of six Yersinia enterocolitica isolates, two were not identified. Only one nonenteric fermenter, a Pasteurella sp., and four nonfermenters (three Pseudomonas sp. and one Centers for Disease Control group Ve-2) were misidentified.     

Rapid differentiation of fermentative from nonfermentative gram-negative bacilli in positive blood cultures by an impedance method

Rapid differentiation of fermentative gram-negative bacilli (fermenters) from nonfermentative gram-negative bacilli (nonfermenters) in positive blood cultures may help physicians to narrow the choice of appropriate antibiotics for empiric treatment. An impedance method for direct differentiation of fermenters from nonfermenters was investigated. The bacterial suspensions (or positive culture broths containing gram-negative bacteria) were inoculated into the module wells of a Bactometer (bioMérieux, Inc., Hazelwood, Mo.) containing 1 ml of Muller-Hinton broth. The inoculated modules were incubated at 35 degrees C, and the change in impedance in each well was continuously monitored. The amount of time required to cause a series of significant deviations from baseline impedance values was defined as the detection time (DT). The percent change of impedance was defined as the change of impedance at the time interval from DT to DT plus 1 h. After testing 857 strains of pure cultures (586 strains of fermenters and 271 strains of nonfermenters), a breakpoint (2.98%) of impedance change was obtained by discriminant analysis. Strains displaying impedance changes of greater than 2.98% were classified as fermenters; the others were classified as nonfermenters. By using this breakpoint, 98.6% (340 of 345) of positive blood cultures containing fermenters and 98% (98 of 100) of positive blood cultures containing nonfermenters were correctly classified. The impedance method was simple, and the results were normally available within 2 to 4 h after direct inoculation of positive blood culture broths.     

Evaluation of the MicroScan rapid neg ID3 panel for identification of Enterobacteriaceae and some common gram-negative nonfermenters

The MicroScan Rapid Neg ID3 panel (Dade Behring, Inc., West Sacramento, Calif.) is designed for the identification of gram-negative bacilli. We evaluated its ability to accurately identify Enterobacteriaceae that are routinely encountered in a clinical laboratory and glucose nonfermenting gram-negative bacilli. Using 511 stock cultures that were maintained at -70 degrees C and passaged three times before use, we inoculated panels according to the manufacturer's instructions and processed them in a Walk/Away instrument using version 22.01 software. The time to identification was 2 h and 30 min. All panel identifications were compared to reference identifications previously determined by conventional tube biochemicals. At the end of the initial 2.5-h incubation period, 405 (79.3%) identifications were correct. An additional 49 (9.6%) isolates were correctly identified after required additional off-line biochemical tests were performed. Thus, at 24 h, 88.8% of the 511 strains tested were correctly identified. Twenty-two (4.3%) were identified to the genus level only. Twenty-six (5.1%) strains were misidentified. Because the system is based on fluorogenics, there are no conventional tests readily available with which to compare possibly incorrect reactions. Of the 28 Salmonella strains that were tested, 5 were incorrectly reported. The 21 remaining errors were scattered among the genera tested. Testing on nine strains gave a result of "no identification" (very rare biotype). The Rapid Neg ID3 panel in this study approached 89% accuracy for the identification of gram-negative organisms encountered in the hospital laboratory.     

Four methods for identification of gram-negative nonfermenting rods: organisms more commonly encountered in clinical specimens.

Four commercial kits, Oxi/Ferm (OF), API 20E (AP), Minitek (MT; BBL Microbiology Systems), and Flow N/F (NF), were evaluated, without additional tests, for identification of 258 gram-negative nonfermentative rods. OF and MT were read after 48 h of incubation, and AP and NF were read after both 24 and 48 h of incubation, respectively. Overall, OF correctly identified 51% of strains, with 46% as part (but not first) of a spectrum of identifications (SI), and 3% incorrect species identification. MT yielded 85% correct identification, with 15% SI. Of 126 glucose-positive strains, or those with greater than or equal to 3 positive AP reactions after 24 h, 60% were correctly identified, with 40% SI; incubation for an additional 24 h raised the rate of correct identification to 99%, with 1% SI. A total of 132 strains yield less than 3 positive AP reactions after 24 h and were identified after 48 h only; of these, 82% were correctly identified, with 17% SI and 1% incorrect species identification. NF correctly identified 79% of cultures after 24 h, with 21% SI; corresponding figures after an additional 24 h of incubation were 80% and 20%, respectively. All four commercial methods show promise; OF is easiest to inoculate, but requires extra tests for optimal identification. AP reliably identifies the majority of clinically important nonfermenters, with fairly good species identification of saccharolytic strains after 24 h. MT yields reliable identification of most nonfermenters and has the advantage of flexibility. NF is easy to inoculate, yields satisfactory identification rates, and may be read after 24 h of incubation.     

Evaluation of MicroScan Rapid Pos Combo panels for identification of staphylococci.

MicroScan Rapid Pos Combo panels (Baxter Diagnostics, Inc., MicroScan, West Sacramento, Calif.) contain substrates conjugated with fluorophores and substrates with a fluorescent pH indicator. AutoSCAn W/A, an automated panel processor equipped with a fluorometer, reads the panels after 2 h of incubation and can identify staphylococci to the species level. We tested 239 strains belonging to 17 species of staphylococci. All the strains were identified by conventional methods (W.E. Kloos and K.H. Schleifer, J. Clin. Microbiol. 1:82-88, 1975) and by the MicroScan Rapid ID system. The system correctly identified 219 (91.6%) strains; nine (3.8%) identification results were probably correct, and six (2.5%) results were incorrect. The system designated five (2.1%) strains as rare biotypes. The automated MicroScan Rapid ID system is useful and reliable in identifying most human isolates of staphylococci encountered in the clinical laboratory.     

Evaluation of the Phoenix 100 ID/AST system and NID panel for identification of Enterobacteriaceae, Vibrionaceae, and commonly isolated nonenteric gram-negative bacilli

The Phoenix 100 ID/AST system (Becton Dickinson Co., Sparks, Md.) is an automated system for the identification and antimicrobial susceptibility testing of bacterial isolates. This system with its negative identification (NID) panel was evaluated for its accuracy in the identification of 507 isolates of the family Enterobacteriaceae, 57 other nonenteric gram-negative isolates that are commonly isolated in clinical microbiology laboratories, and 138 isolates of the family Vibrionaceae. All of the isolates had been characterized by using approximately 48 conventional tube biochemicals. Of the 507 isolates of the Enterobacteriaceae, 456 (89.9%) were correctly identified to the genus and species levels. The five isolates of Proteus penneri required an off-line indole test, as suggested by the system to differentiate them from Proteus vulgaris. The identifications of 20 (3.9%) isolates were correct to the genus level but incorrect at the species level. Two (0.4%) isolates were reported as "no identification." Misidentifications to the genus and species levels occurred for 29 (5.7%) isolates of the Enterobacteriaceae. These incorrect identifications were spread over 14 different genera. The most common error was the misidentification of Salmonella species. The shortest time for a correct identification was 2 h 8 min. The longest time was 12 h 27 min, for the identification of a Serratia marcescens isolate. Of the 57 isolates of nonenteric gram-negative bacilli (Acinetobacter, Aeromonas, Burkholderia, Plesiomonas, Pseudomonas, and Stenotrophomonas spp.), 48 (84.2%) were correctly identified to the genus and species levels and 7 (12.3%) were correctly identified to the genus level but not to the species level. The average time for a correct identification was 5 h 11 min. Of the Vibrionaceae spp., 123 (89.1%) were correctly identified at the end of the initial incubation period, which averaged 4 h. Based on the findings of this study, the Phoenix 100 ID/AST system NID panel falls short of being an acceptable new method for the identification of the Enterobacteriaceae, Vibrionaceae, and gram-negative nonenteric isolates that are commonly encountered in many hospital microbiology laboratories.     

Evaluation of the Titertek-NF system for identification of gram-negative nonfermentative and oxidase-positive fermentative bacteria

The Titertek-NF (TT-NF) system (Flow Laboratories GmbH, Meckenheim, Federal Republic of Germany) was evaluated for the identification of 1,289 strains of gram-negative, nonfermentative bacteria and some gram-negative, oxidase-positive bacteria. The oxidase test was also performed. Identifications were classified as correct, not identified (two or more taxa possible and identification score of less than 80%; supplementary tests for furthering the identification were not performed), and incorrect. Correct identification results were further subdivided by the correct level of species or biotype identification as greater than or equal to 98% (category a), 90 to 97% (category b), and 80 to 89% (category c). When compared with conventional identification results, the TT-NF system correctly identified 90.3% of strains (1,164 of 1,289 strains), with 72.5% (935 strains) belonging to category a, 14.7% of strains (189 strains) belonging to category b, and 3.1% of strains (40 strains) belonging to category c. Among the remaining strains, 104 (8.1%) were not identified and 14 (1.1%) were misidentified, and the system failed to generate identification results for 7 strains (0.5%). Reactions within the TT-NF system were reproducible, with an estimated probability of erroneous test results of 0.2%.     

Preliminary evaluation of Biolog, a carbon source utilization method for bacterial identification.

The Biolog Identification System (Biolog, Inc., Hayward, Calif.) is a new bacterial identification method that establishes an identification based on the exchange of electrons generated during respiration, leading to a subsequent tetrazolium-based color change. This system tests the ability of a microorganism to oxidize a panel of 95 different carbon sources. We report on a preliminary investigation of the ability of the instrument to identify, using its computer-driven enzyme immunoassay reader, a diverse group of clinically relevant members of the family Enterobacteriaceae and gram-negative non-Enterobacteriaceae. The Biolog reported identifications (correct or incorrect) for 266 of 352 organisms tested (75.6%). Of the 266 identifications reported, 87.3% were correct at the genus level and 75.6% were correct at the species level at 24 h. In the total study of 352 strains, 46.6% were correct to the species level at 4 h and 57.1% were correct to the species level at 24 h. The error rate was 10.4% after 4 h and 9.6% after 24 h. The Biolog performed well with many genera, but problems were encountered with some strains of Klebsiella, Enterobacter, and Serratia. We found the system to be versatile and easy to use.     

Evaluation of the autoSCAN-W/A system for rapid (2-hour) identification of members of the family Enterobacteriaceae.

We evaluated the ability of the Baxter autoSCAN-W/A System (MicroScan Division, Baxter Diagnostics, Inc., West Sacramento, Calif.) to use the rapid (2-h) gram-negative identification panel for accurate identification of members of the family Enterobacteriaceae. At 2 h, 353 of 467 (75.6%) strains in a challenge set of biochemically typical and atypical stock cultures were correctly identified to genus and species. Another 76 (16.3%) strains were correctly identified to genus and species after the performance of recommended additional biochemical testing. Thus, at 24 h, 91.9% of the 467 strains were correctly identified. Twenty-two strains (4.7%) were identified to the correct genus but the incorrect species, and 16 strains (3.4%) were misidentified. Of these 16 strains, 9 were incorrect at 2 h, and 7 were incorrect after the additional testing. Because the system is based on fluorogenic substrates, no conventional tests were readily available with which to compare aberrant reactions. These results suggest that the autoSCAN-W/A with its rapid gram-negative panels is acceptable for the identification of the Enterobacteriaceae in a clinical microbiology laboratory.     

Identification of Gram-negative non-fermenters and oxidase-positive fermenters by the Oxi/Ferm Tube

Since the recent introduction of the Roche Oxi/Ferm Tube to the UK two identification schemes have been developed by the manufacturer for use with the kit. We evaluated the success of these two schemes in identifying 222 predominantly culture collection strains belonging to 45 taxa of non-fermenters and nine taxa of oxidase-positive fermenters. The strains were chosen to represent all the taxa included in the two identification schemes developed by the manufacturer and we have therefore been able to assess the overall success of identification by the two schemes. Since, however, our choice of strains does not reflect their incidence in clinical material, our identification rates are not necessarily those that might be obtained in a routine clinical laboratory. The most advanced identification scheme so far developed for the Oxi/Ferm Tube (CCIS System 1977-1432) allowed 62% of the 222 strains to be correctly identified although a disturbing feature was that more of the strains that were not correctly identified were incorrectly identified (24%) rather than not identified (14%); these figures represent an improvement over the earlier identification scheme (CCIS System 1976-621-74346) for which the corresponding figures were 56%, 32%, and 12%. CCIS System 1977-1432 seems likely to give a better performance in a routine clinical laboratory than in this study since for those taxa which, we would judge from the material sent to us for identification, are most commonly seen in a routine laboratory (Acinetobacter calcoaceticus, A. lwoffii, Pseudomonas aeruginosa, P. fluorescens, P. maltophilia, P. pseudoalcaligenes, and P. putida) 89% were correctly identified, none remained unidentified, and 11% were incorrectly identified. Thirty strains, each of a different taxon, were tested in triplicate to assess the reproducibility of reactions in the Oxi/Ferm Tube.     

Evaluation of commercial test systems for the identification of nonfermenters

Three commercially available test systems for the identification of nonfermentative gramnegative rods were compared: API 20NE, Flow N/F and Minitek Nonfermenter System. Two hundred strains were identified by conventional means and by each test system. The rate of correct identification ofAcinetobacter, Alcaligenes, Flavobacteriwn andMoraxella strains to genus level and of the other genera to species level was 92 % with API 20NE, 84 % with flow N/F and 75 % with Minitek Nonfermenter System. The need for these kits in the diagnostic hospital laboratory is also discussed.     

Rapid Identification of Bacteria and Yeasts from Positive-Blood-Culture Bottles by Using a Lysis-Filtration Method and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum Analysis with the SARAMIS Database

Rapid identification of microorganisms causing bloodstream infections directly from a positive blood culture would decrease the time to directed antimicrobial therapy and greatly improve patient care. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable method for identifying microorganisms from positive culture. This study evaluates the performance of a novel filtration-based method for processing positive-blood-culture broth for immediate identification of microorganisms by MALDI-TOF with a Vitek MS research-use-only system (VMS). BacT/Alert non-charcoal-based blood culture bottles that were flagged positive by the BacT/Alert 3D system were included. An aliquot of positive-blood-culture broth was incubated with lysis buffer for 2 to 4 min at room temperature, the resulting lysate was filtered through a membrane, and harvested microorganisms were identified by VMS. Of the 259 bottles included in the study, VMS identified the organisms in 189 (73%) cultures to the species level and 51 (19.7%) gave no identification (ID), while 6 (2.3%) gave identifications that were considered incorrect. Among 131 monomicrobic isolates from positive-blood-culture bottles with one spot having a score of 99.9%, the IDs for 131 (100%) were correct to the species level. In 202 bottles where VMS was able to generate an ID, the IDs for 189 (93.6%) were correct to the species level, whereas the IDs provided for 7 isolates (3.5%) were incorrect. In conclusion, this method does not require centrifugation and produces a clean spectrum for VMS analysis in less than 15 min. This study demonstrates the effectiveness of the new lysis-filtration method for identifying microorganisms directly from positive-blood-culture bottles in a clinical setting.     

Evaluation of Micro-Media Quad Panels for identification of the Enterobacteriaceae.

The Micro-Media Quad Enteric Panel and coding system were evaluated by comparing the identifications of 193 stock and 216 clinical isolates, representing 31 species of Enterobacteriaceae, with those provided by conventional methods. The results corresponded for 91% of the organisms if only the single or most probable identification was accepted and for 93% if the correct identification was listed as one of several possible, but not the most probable, identification by the coding system. With 73% of the isolates, the single identification provided by the coding system was correct, and in 20% of instances the correct identification was listed as the first of several choices in descending order of probability but requiring additional tests for differentiation. In most of the latter instances, these additional tests consisted of serological confirmation of salmonellae or shigellae. Discrepancies consisted of incorrect identifications (4%) and organisms with code numbers not appearing in the coding system (3%).     

Comparison of Rapid NFT system and conventional methods for identification of nonsaccharolytic gram-negative bacteria

This study examined the Rapid NFT system (Analytab Products, Plainview, N.Y.) to determine its ability to accurately identify 229 clinical isolates of mostly nonsaccharolytic gram-negative rods. Identifications were classified by the following scheme: correct (corresponding to excellent, very good, good, or acceptable identification as listed in the code book); low discrimination (correct identification among a range of listed possibilities, with additional tests necessary for accurate identification); incorrect. Correct identification was considered correct to species and subspecies for all organisms except Alcaligenes faecalis and "Alcaligenes odorans"; "A. faecalis/odorans" was considered a correct response. By using these criteria, 71.6% of the strains were correctly identified, 17.9% were identified with low discrimination, and 10.5% were incorrectly identified. When consideration was made for incorrect identification resulting from taxonomic problems (e.g., Alcaligenes and Moraxella spp.), incorrect identifications fell to 5.2%. The Rapid NFT system was truly rapid and was easy to use and interpret. Its use of carbon substrate assimilation enables it to provide more accurate identification of medically important nonsaccharolytic bacteria than do other commercially available systems.     

Evaluation of two miniaturized systems, MicroScan W/A and BBL Crystal E/NF, for identification of clinical isolates of Aeromonas spp

Fifty-two clinical strains and 22 type and reference Aeromonas strains, previously genetically characterized by 16S rRNA gene restriction fragment length polymorphism, were identified in parallel with the MicroScan Walk/Away and BBL Crystal Enteric/Nonfermenter systems. The former identified only 14.8% of the isolates correctly, and the latter identified only 20.3% correctly, which indicates that neither of these systems is useful for this purpose.