Multi-Laboratory Evaluation of an Automated Microbial Detection/Identification System

An automated and computerized system (Automicrobic System [AMS]) for the detection of frequently encountered bacteria in clinical urine specimens was tested in a collaborative study among six laboratories. The sensitivity, specificity, reliability, and reproducibility of the AMS were determined, and the system was compared with conventional detection and identification systems. In this study, pure cultures and mixtures of pure cultures were used to simulate clinical urine specimens. With pure cultures, the sensitivity of the AMS in identifying the nine groups of organisms most commonly found in urine averaged 92.8%. The specificity averaged 99.4%, and the reliability of a positive result averaged 92.1%. The latter value was strongly influenced by a relatively high occurrence of false positive Escherichia coli results. The AMS was capable of detecting growth of most organisms, including those which it was not designed to identify. However, it identified some of these incorrectly as common urinary tract flora. Reproducibility of results, both within laboratories and among different laboratories, was high. Fast-growing organisms, such as E. coli and Klebsiella/Enterobacter species, were detected often at cell populations well below the AMS enumeration threshold of 70,000/ml. In mixed culture studies, high levels of sensitivity and specificity were maintained but when Serratia species were present in mixtures with other organisms, there was often a false positive report of E. coli. The overall performance of the AMS was considered satisfactory under the test conditions used.     

Capillary Electrophoresis–Single-Strand Conformation Polymorphism Analysis for Rapid Identification of Pseudomonas aeruginosa and Other Gram-Negative Nonfermenting Bacilli Recovered from Patients with Cystic Fibrosis

We used capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF). Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes. The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer. Data were analyzed with GeneScan 672 software. This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers. Thirty-four reference strains belonging to the genera Pseudomonas, Brevundimonas, Burkholderia, Comamonas, Ralstonia, Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism. The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (Escherichia coli 16S rRNA positions 1173 to 1389 [M. N. Widjojoatmodjo, A. C. Fluit, and J. Verhoef, J. Clin. Microbiol. 32:3002-3007, 1994]). This primer set differentiated the main CF pathogens from closely related species but did not distinguish P. aeruginosa from Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcaligenes denitrificans. Two hundred seven CF clinical isolates (153 of P. aeruginosa, 26 of Stenotrophomonas maltophilia, 15 of Burkholderia spp., and 13 of A. xylosoxidans) were tested with P11P-P13P. The CE-SSCP patterns obtained were identical to those for the corresponding reference strains. Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains. This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.     

VITEK-2 compact全自动微生物鉴定仪对葡萄球菌鉴定能力的评价

目的评价VITEK-2 compact全自动微生物鉴定仪对葡萄球菌的鉴定能力。方法收集从我院病人标本中分离的葡萄球菌81株。常规细菌培养后,用VITEK-2 compact和API Staph系统进行检测,以API Staph系统为参照,评价VITEK-2 compact的优势和不足。结果 VITEK-2 compact和API Staph系统的总体鉴定符合率为95.1%,其中金黄色葡萄球菌的鉴定符合率为100%,凝固酶阴性葡萄球菌的鉴定符合率为90.5%。结论 VITEK-2 compact鉴定系统能够满足临床工作的需求,其中对金黄色葡萄球菌的鉴定率较高,在进行凝固酶阴性葡萄球菌鉴定时有一定的局限性,需要其他方法予以补充。     

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.     

Development and Evaluation of an Automated Annotation Pipeline and cDNA Annotation System

Manual curation has long been held to be the “gold standard” for functional annotation of DNA sequence. Our experience with the annotation of more than 20,000 full-length cDNA sequences revealed problems with this approach, including inaccurate and inconsistent assignment of gene names, as well as many good assignments that were difficult to reproduce using only computational methods. For the FANTOM2 annotation of more than 60,000 cDNA clones, we developed a number of methods and tools to circumvent some of these problems, including an automated annotation pipeline that provides high-quality preliminary annotation for each sequence by introducing an “uninformative filter” that eliminates uninformative annotations, controlled vocabularies to accurately reflect both the functional assignments and the evidence supporting them, and a highly refined, Web-based manual annotation tool that allows users to view a wide array of sequence analyses and to assign gene names and putative functions using a consistent nomenclature. The ultimate utility of our approach is reflected in the low rate of reassignment of automated assignments by manual curation. Based on these results, we propose a new standard for large-scale annotation, in which the initial automated annotations are manually investigated and then computational methods are iteratively modified and improved based on the results of manual curation.     

Evaluation of the Vitek 2 System for Rapid Identification of Clinical Isolates of Gram-Negative Bacilli and Members of the Family Streptococcaceae

Accuracy of the Vitek 2 automated system (bioMérieux Vitek, USA) for rapid identification of bacteria was evaluated using a collection of 858 epidemiologically unrelated gram-negative and 99 gram-positive clinical isolates. Isolates were tested after subculturing to ensure purity. Conventional agar-based biochemical tests (Steers replicator) were used as a reference method of identification. Gram-negative bacteria were identified to the species level with 95.3% accuracy by the system (Enterobacteriaceae, 95.9%; and non-Enterobacteriaceae, 92.5%), and gram-positive isolates with 72% accuracy. Although Vitek 2 identified routine clinical isolates of gram-negative bacilli and Enterococcus faecalis and Enterococcus faecium reliably, rapidly, and reproducibly, improvement is required in the identification of less common species of enterococci and viridans group streptococci. Electronic Publication     

Preprototype of an Automated Microbial Detection and Identification System: a Developmental Investigation

The AutoMicrobic System is an automated, computerized instrument that uses highly selective media and an optical system for detection, enumeration, and identification of bacteria and some yeasts in 13 h. A preprototype instrument (AutoMicrobic System-1) and its urine culture kit (Identi-Pak), developed for the detection, enumeration, and identification of eight species or groups of bacteria and of Candida species and Torulopsis glabrata in urine specimens, was evaluated during its development. An overall agreement of approximately 90% between the preprototype instrument and conventional (manual) culture methods has been obtained both with 1,473 seeded (simulated) and 1,688 clinical (mono- or polymicrobial) specimens containing 70,000 (or more) colony-forming units per ml of Escherichia coli, Klebsiella-Enterobacter species, Proteus species, Citrobacter freundii, Serratia species, group D enterococci, or yeasts (Candida species and T. glabrata). Lower agreements in identification were obtained with Pseudomonas aeruginosa-containing (average of 75% in clinical specimens) and Staphylococcus aureus-containing (76%) specimens. Comparison of specimens tested simultaneously in two preprototype systems resulted in =/<4% disagreement; true negativity agreements in all specimen groups tested were at least 94%. Among problems remaining are adaptation of system for specimens other than urine, improvement of sensitivity for P. aeruginosa and S. aureus, and standardization of manual methods used for comparison and validation.     

Automated Microbiological Detection/Identification System

An automated, computerized system, the AutoMicrobic System, has been developed for the detection, enumeration, and identification of bacteria and yeasts in clinical specimens. The biological basis for the system resides in lyophilized, highly selective and specific media enclosed in wells of a disposable plastic cuvette; introduction of a suitable specimen rehydrates and inoculates the media in the wells. An automated optical system monitors, and the computer interprets, changes in the media, with enumeration and identification results automatically obtained in 13 h. Sixteen different selective media were developed and tested with a variety of seeded (simulated) and clinical specimens. The AutoMicrobic System has been extensively tested with urine specimens, using a urine test kit (Identi-Pak) that contains selective media for Escherichia coli, Proteus species, Pseudomonas aeruginosa, Klebsiella-Enterobacter species, Serratia species, Citrobacter freundii, group D enterococci, Staphylococcus aureus, and yeasts (Candida species and Torulopsis glabrata). The system has been tested with 3,370 seeded urine specimens and 1,486 clinical urines. Agreement with simultaneous conventional (manual) cultures, at levels of 70,000 colony-forming units per ml (or more), was 92% or better for seeded specimens; clinical specimens yielded results of 93% or better for all organisms except P. aeruginosa, where agreement was 86%. System expansion in progress includes antibiotic susceptibility testing and compatibility with most types of clinical specimens.     

Evaluation of a Dual-Staining Method for Acid-Fast Bacilli

A dual-staining procedure for acid-fast bacilli was found to have poor correlation with the Ziehl-Neelsen and auramine-rhodamine staining techniques.     

Identification and screening of carbapenemase-producing Enterobacteriaceae

Carbapenem-hydrolysing β-lactamases are the most powerful β-lactamases, being able to hydrolyse almost all β-lactams. They are mostly of the KPC, VIM, IMP, NDM and OXA-48 types. Their current extensive spread worldwide in Enterobacteriaceae is an important source of concern, as these carbapenemase producers are multidrug-resistant. Detection of infected patients and of carriers are the two main approaches for prevention of their spread. Phenotypic and molecular-based techniques are able to identify these carbapenemase producers, although with variable efficiencies. The detection of carriers still relies mostly on the use of screening culture media.     

Evaluation of the BD Phoenix Automated Identification and Susceptibility Testing System in Clinical Microbiology Laboratory Practice

The Phoenix Automated Microbiology System (BD Biosciences, USA) is a new, fully automated system for the rapid identification and antimicrobial susceptibility testing of gram-positive and gram-negative bacteria. The objective of this study was to evaluate the quality of performance of the Phoenix system in the identification and antimicrobial susceptibility testing of 260 gram-negative (n=174) and gram-positive (n=86) isolates collected from Polish hospitals in recent years. Two Phoenix panel types for identification/antimicrobial susceptibility testing, NMIC/ID-5 for gram-negative rods and PMIC/ID-4 for gram-positive cocci, were used in the analysis according to the manufacturer's recommendations. The results produced by the system were compared with data obtained by reference or conventional microbiological methods. A high rate of agreement between the Phoenix and the conventional methods was observed for identification, ranging from 100% for gram-positive cocci to 96.0% for gram-negative nonfermenters and 92.5% for members of the family Enterobacteriaceae. Similarly, a high level of agreement characterized the antimicrobial susceptibility data obtained with the Phoenix and by the agar dilution method (2,361 test results). For staphylococci, enterococci and Enterobacteriaceae, the methods were 100% concordant in determining the category of susceptibility of isolates to the majority of the antimicrobial agents tested. A category agreement value of below 90% was found for the susceptibility of enterococci and gram-negative nonfermenters to ciprofloxacin (84.6% and 88.5%, respectively) and for susceptibility of Stenotrophomonas maltophilia to trimethoprim-sulfamethoxazole (80.0%).     

Validation of Vitek 2 Nonfermenting Gram-Negative Cards and Vitek 2 Version 4.02 Software for Identification and Antimicrobial Susceptibility Testing of Nonfermenting Gram-Negative Rods from Patients with Cystic Fibrosis

Accurate identification and antimicrobial susceptibility testing (AST) of nonfermenters from cystic fibrosis patients are essential for appropriate antimicrobial treatment. This study examined the ability of the newly designed Vitek 2 nonfermenting gram-negative card (NGNC) (new gram-negative identification card; bioMérieux, Marcy-l''Ètoile, France) to identify nonfermenting gram-negative rods from cystic fibrosis patients in comparison to reference methods and the accuracy of the new Vitek 2 version 4.02 software for AST compared to the broth microdilution method. Two hundred twenty-four strains for identification and 138 strains for AST were investigated. The Vitek 2 NGNC identified 211 (94.1%) of the nonfermenters correctly. Among morphologically atypical microorganisms, five strains were misidentified and eight strains were determined with low discrimination, requiring additional tests which raised the correct identification rate to 97.8%. Regarding AST, the overall essential agreement of Vitek 2 was 97.6%, and the overall categorical agreement was 92.9%. Minor errors were found in 5.1% of strains, and major and very major errors were found in 1.6% and 0.3% of strains, respectively. In conclusion, the Vitek NGNC appears to be a reliable method for identification of morphologically typical nonfermenters and is an improvement over the API NE system and the Vitek 2 GNC database version 4.01. However, classification in morphologically atypical nonfermenters must be interpreted with care to avoid misidentification. Moreover, the new Vitek 2 version 4.02 software showed good results for AST and is suitable for routine clinical use. More work is needed for the reliable testing of strains whose MICs are close to the breakpoints.Pseudomonas aeruginosa is the most important cause of lung infections in patients with cystic fibrosis (CF) (14). In our hospital, 50% of sputum-producing CF patients are colonized in their lower airways with P. aeruginosa or other nonfermenting bacteria. Accurate identification and antimicrobial susceptibility testing (AST) are essential for appropriate antimicrobial therapy.A variety of automated commercial systems for identification and susceptibility testing of nonfermenting bacteria are available (2, 3, 12, 19, 20). They are widely used because of the increasing volumes of clinical specimens processed by clinical laboratories and perceived cost effectiveness. The automated systems decrease the in-laboratory turnaround time and enable a faster targeted antimicrobial therapy. Unfortunately, errors in classification and AST by any test system can have serious implications for the clinical outcome of patients. The most frequently reported errors have involved the inaccurate identification of nonfermenters due to their phenotypic variations and slower growth rates and the inconsistencies between the tested broad-spectrum β-lactam antibiotics. Because of the perceived inaccuracies of AST from CF isolates, a consensus conference on CF microbiology recommended the use of the disk diffusion method for testing P. aeruginosa and other nonfermenters (13, 22).To improve the identification rate of nonfermenting gram-negative bacilli, a new colorimetric Vitek 2 card (nonfermenting gram-negative card [NGNC]) with an enlarged database was recently introduced. To advance the accuracy of the AST results, a new software (version 4.02) was developed.The aim of the present study was to evaluate the performance of the new NGNC for identification and the new software version 4.02 for AST of nonfermenters isolated from CF patients.     

Clinical Evaluation of the MICRO-ID, API 20E, and Conventional Media Systems for Identification of Enterobacteriaceae

MICRO-ID (General Diagnostics, Morris Plains, N.J.) is a new kit system designed for the identification of Enterobacteriaceae in 4 h. It consists of 15 biochemical tests of paper disks. Each test is in its own compartment in a molded plastic tray. Only one reagent need be added to the system (2 drops of 20% KOH, which is added to the Voges-Proskauer test). Based on the pattern of positive and negative biochemical test results, a five-digit octal code number is calculated. An identification is derived from a computer-generated identification manual. A study was conducted to compare three systems-the MICRO-ID 4-h and the API 20E (Analytab Products Inc., Plainview, N.Y.) 18- to 24-h systems and a conventional media system-to measure the ability of each to identify members of the family Enterobacteriaceae. Comparison tables, rather than simple percentage agreement tables, were generated to define the particular strengths and weaknesses of each system and allow the laboratory to best use the data. The MICRO-ID compared quite favorably with conventional media. MICRO-ID yielded incorrect identifications with 1.5% of the isolates tested (API 20E, 4.7% misidentification rate). Half the MICRO-ID misidentifications occurred when the system identified a Citrobacter diversus as a lysine-negative Escherichia coli; all gave one octal number. A direct comparison of the MICRO-ID and API 20E was of limited value because percentage agreements were merely the sums of the errors of each. The ease of inoculation, the requirement for the addition of only one reagent, and the 4-h capability make the MICRO-ID system an extremely attractive development in the field of bacterial identification.     

Evaluation of an Automated Instrument for Inoculating and Spreading Samples onto Agar Plates

The findings from a preliminary assessment of a new instrument designed for the inoculation and spreading of specimens for microbiological analysis onto agar plates are described. The study found that the instrument was able to select full or biplates from a number of input cassettes, each containing different agar types. Samples were then inoculated by the instrument onto the agar surfaces and spread by a novel plastic applicator. Following this, the instrument labeled the plates and sorted them into a number of specified output stations. It was found that the instrument was able to inoculate and spread samples over a greater proportion of the agar plate surface than the manual loop-to-plate method. As a consequence, up to 44% more usable colonies were produced per plate from clinical specimens and standard cultures. Viable counts showed that the instrument was able to detect as few as 10(2) CFU/ml in fluids and also facilitated the enumeration of organisms, particularly in specimens such as urine.     

Evaluation of the Liaison Automated Testing System for Diagnosis of Congenital Toxoplasmosis

Congenital toxoplasmosis is a worldwide health problem, and different screening strategies exist. Testing of toxoplasma-specific antibodies in infants identifies congenital toxoplasmosis during the first year of life. However, experience with commercial available immunoassays is limited. The aim of this study was to evaluate both the performance and analytical characteristics of the Liaison diagnostic system in infants. In a retrospective study, serum Toxoplasma gondii antibodies were measured in samples from 333 infants, including 212 noninfected infants and 121 infants with congenital toxoplasmosis. A total of 1,157 umbilical cord blood and peripheral serum samples were analyzed. Liaison toxoplasma-specific IgG and IgM antibodies and the IgG avidity index were compared to the infection status of the infant, determined by the Sabin-Feldman dye test and immunosorbent agglutination assay—IgM. All noninfected infants were seronegative by Liaison IgG within the first year of life. The Liaison system showed a sensitivity of 81.8%, a specificity of 100.0%, a positive predictive value of 100.0%, a negative predictive value of 90.6%, and overall agreement of 84.4% by comparison with the dye test. Overall agreement of both IgM test systems was 96.0%. In this study cohort, avidity did not show a potential diagnostic benefit for the detection of congenital infection. In conclusion, the Liaison system is a valuable tool to monitor the serologic course of infants at risk. A final serologic confirmatory test is recommended to improve the rate of detection of congenital toxoplasmosis at 1 year of life. Protocols of routine follow-up testing in infants and accurate diagnostic tools after acute gestational infections are needed to improve medical care.     

Evaluation of an automated, computerized system (automicrobic system) for Enterobacteriaceae identification.

The automated and computerized AutoMicrobic system (AMS; Vitek Systems, Inc., subsidiary of McDonnell Douglas, Hazelwood, Mo.) was evaluated as a means of identifying the Enterobacteriaceae. The Micro-ID system (General Diagnostics, Morris Plains, N.J.) and, when necessary, conventional tubed media were used for comparison. Identification by AMS and Micro-ID differed in only 12 of 1,528 isolates (0.8%). Disagreements occurred primarily with Enterobacter spp. Precision testing of the AMS showed only 1 of 72 tests (1.4%) deviating from the expected. The AMS was found to be an accurate and precise method for the identification of Enterobacteriaceae.