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Identification of concurrent harmonic and inharmonic vowels: a test of the theory of harmonic cancellation and enhancement.

The improvement of identification accuracy of concurrent vowels with differences in fundamental frequency (delta F0) is usually attributed to mechanisms that exploit harmonic structure. To decide whether identification is aided primarily by selecting the target vowel on the basis of its harmonic structure ("harmonic enhancement") or removing the interfering vowel on the basis of its harmonic structure ("harmonic cancellation"), pairs of synthetic vowels, each of which was either harmonic or inharmonic, were presented to listeners for identification. Responses for each vowel were scored according to the vowel's harmonicity and that of the vowel that accompanied it. For a given target, identification was better by about 3% for a harmonic ground unless the target was also harmonic with the same F0. This supports the cancellation hypothesis. Identification was worse for harmonic than for inharmonic targets by 3%-8%. This does not support the enhancement hypothesis. When both vowels were harmonic, identification was better by about 6% when the F0's differed by 1/2 semitone, consistent with previous experiments. Results are interpreted in terms of harmonic enhancement and harmonic cancellation, and alternative explanations such as waveform interaction are considered.

Adult↗

Evaluation of the Minitek system for direct identification of anaerobic rods from positive blood cultures.

The direct inoculation of the Minitek anaerobe identification system (BBL Microbiology Systems, Cockeysville, Md.) from positive blood cultures was compared with subculture and Minitek results obtained using the manufacturer's recommended procedures. A total of 40 clinical anaerobic blood cultures were processed for rapid identification utilizing bacterial pellets obtained by centrifugation. Of these cultures, 30 yielded pure isolates of anaerobic rods that were used for comparison. In 87% of the pure cultures, identification from the direct inoculum method was identical to the routine procedure using Minitek biochemicals. When the additional test for lecithinase and lipase production was included, the identification agreement was 97%. Direct identification of anaerobic rods from blood cultures utilizing the Minitek system is reliable, easy to perform, and can provide a complete identification in 24 h from the time a blood culture showed growth.

Anaerobiosis↗

Evaluation of the updated MS-2 Bacterial Identification system in comparison with the API 20E system.

The recently updated MS-2 Bacterial Identification system software (Abbott Laboratories, Diagnostic Division, Irving, Tex.) was compared with the original MS-2 Bacterial Identification software and the API 20E, using 968 strains of Enterobacteriaceae. The updated MS-2 software correctly identified 94.4% of the isolates tested. API 20E and the original MS-2 software correctly identified 91 and 85.3% of the strains, respectively. MS-2 responses were considered to be equivocal (needing additional tests for verification) if the percent likelihood values were less than 80%. The percentage of equivocal responses was reduced from 6.5% with the original software to 2.2% with the updated software, and the percentage of incorrect identifications was reduced from 8.2 to 3.4% with the original and updated software, respectively. Organisms belonging to 25 taxonomic groups were tested. Direct comparison of the two MS-2 programs showed that the updated MS-2 software increased the identification accuracy of Salmonella spp., Enterobacter cloacae, Providencia stuartii, Escherichia coli, Shigella spp., Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, and Acinetobacter calcoaceticus. A decrease in accuracy was seen with Citrobacter freundii, Hafnia alvei, Enterobacter agglomerans, and Yersinia pseudotuberculosis when the updated software was used. The remaining 12 taxonomic groups were not affected by the software changes. The updated MS-2 software appears to significantly improve the identification accuracy of the MS-2 Bacterial Identification system.

Autoanalysis↗

Evaluation of the rapid NFT system for identification of gram-negative, nonfermenting rods.

This study evaluated the ability of the Rapid NFT system (API System SA, Montalieu-Vercieu, France) to accurately identify 262 clinically isolated, gram-negative, nonfermentative rods without additional tests. Identifications were classified as correct; low discrimination, with a spectrum of two or more possibilities (additional tests necessary for accurate identification); and incorrect. Correct identification rates were analyzed in two categories: (i) correct to species or biotype for all organism groups except Alcaligenes faecalis-odorans, Moraxella, Pseudomonas testosteroni-alcaligenes-pseudoalcaligenes, and Acinetobacter calcoaceticus biotype haemolyticus-alcaligenes (in this category, the latter four genus-biotype group identifications were taken as correct) and (ii) correct to species or biotype in all cases, including the above four groups. In category i, 87.4% of the strains were correctly identified, with 4.2% low discrimination and 8.4% incorrect. When the criteria of category ii were used, 71.8% of the strains were correctly identified, with 19.9% low discrimination. The Rapid NFT system provided excellent species identification of Pseudomonas and Flavobacterium spp., Bordetella bronchiseptica, and Achromobacter xylosoxidans strains. Within Acinetobacter calcoaceticus, differentiation between biotypes anitratus and lwoffi was satisfactory, but the system did not differentiate between biotypes haemolyticus and alcaligenes. Species resolution within the genera Moraxella and Alcaligenes was incomplete. All Alcaligenes faecalis strains were misidentified and accounted for 50% of misidentifications with the Rapid NFT system; however, these results may reflect taxonomic differences rather than true misidentifications. The Rapid NFT system is easy to inoculate and interpret and represents a worthwhile advance in the identification of gram-negative, nonfermentative rods.

Bacteria↗

Clinical evaluation of the Vitek ANI card for identification of anaerobic bacteria.

An evaluation of the Vitek Anaerobe Identification (ANI) card was performed with 341 bacterial isolates, including 313 clinical isolates and 28 stock strains of anaerobic microorganisms. Identifications obtained with the ANI card were compared with those determined by conventional methods. The card identified 73.2% of 149 anaerobic gram-negative bacilli, 63.6% of 44 Clostridium spp., 65.8% of 38 anaerobic nonsporeforming gram-positive bacilli, and 69.1% of 110 anaerobic cocci, with no further testing required. When genus-level identifications were included, 83.9% of the anaerobic gram-negative bacilli, 70.5% of Clostridium spp., 73.7% of the anaerobic nonsporeforming gram-positive bacilli, and 73.6% of the anaerobic cocci were identified. Nineteen isolates (5.6%) produced identifications of good confidence but marginal separation or questionable biotype, in which the correct identification was listed with one or two other possible choices and extra tests were required and suggested. A total of 28 (8.2%) were not identified and 29 isolates (8.5%) were misidentified by the ANI card. Among the commonly isolated clinically significant anaerobes, the ANI card identified 100% of 55 Bacteroides fragilis and 100% of 8 Clostridium perfringens. Use of supplemental tests and expansion of the data base to include additional strains of organisms that are difficult to separate even with conventional methods may improve the accuracy of the ANI card as a method for identification of anaerobic bacteria in the clinical laboratory.

Bacteria, Anaerobic↗

T-mod pathway, a reduced sequence for identification of gram-negative urinary tract pathogens.

In this paper, we describe a reduced sequence of identification that includes T-mod medium, a selective and differential isolation medium which allows accurate presumptive identification of the most common gram-negative bacteria encountered in urine samples. The present study, performed on bacteria isolated from 1,762 independent urine samples, has shown that a few selected tests (lysine and ornithine decarboxylase, urease and trehalose fermentation tests) improve the identification accuracy of T-mod, making it possible both to identify the less frequent species and to prevent some misidentifications of Klebsiella pneumoniae and Proteus mirabilis. The proposed work flow agreed with conventional identification protocols to a 99.3% extent and allowed identification of 87.4% of the isolates directly from the primary plate, 11.4% after 1 to 3 additional tests, and 1.2% after an identification gallery.

Bacteriuria↗

Accuracy and reproducibility of the MicroScan rapid anaerobe identification system with an automated reader.

Rapid anaerobe identification (MicroScan) panels (4 h) were evaluated both visually and by the AutoScan-4, a computer-controlled microplate reader. The results of both reading methods were compared with identifications obtained by the conventional (Virginia Polytechnic Institute) method. In total, 237 anaerobes were tested. Correct identifications were obtained for 166 strains (70%) by visual reading and 157 strains (66.2%) by the AutoScan-4. Supplementary tests resulted in 80.1 and 76.7% total correct identifications, respectively. Comparison of the two reading methods revealed complete agreement for 169 strains. Differences between the two reading methods were due to difficulties in reading specific reactions. This was especially true with the clostridial species. The performance of the MicroScan system in the identification of anaerobic bacteria appears comparable to that of other 4-h identification systems for anaerobes, but this system shows significant variance from the conventional system. Improvements in the trays and data base are required before the system can be recommended for routine use.

Autoanalysis↗

Evaluation of autoSCAN-W/A automated microbiology system for the identification of non-glucose-fermenting gram-negative bacilli.

We evaluated the ability of the autoSCAN-W/A (MicroScan Division, Baxter Healthcare Corporation, West Sacramento, Calif.), in conjunction with the dried colorimetric Neg ID type 2 panel (DCP) and new rapid fluorometric Neg ID panel (RFP), to identify non-glucose-fermenting gram-negative bacilli by challenging the system with 310 previously identified reference strains. Of these 310 isolates, 286 organisms were in the DCP data base and 269 were in the RFP data base. Use of the DCP panels resulted in 118 (41.3%) correct and 64 (22.4%) incorrect first choice identifications at greater than or equal to 85% probability, 61 (21.3%) low-probability identifications, and 43 (15.0%) reports of unidentified organisms. The RFP system reported 135 (50.1%) correct and 25 (9.3%) incorrect identifications at greater than or equal to 85% probability and 109 (40.5%) low-probability identifications. Unidentified isolates (DCP system only) and isolates producing low-probability first choice identifications (both systems) required supplementary biochemical testing. Over half (37 of 64 [57.8%]) of the DCP misidentifications were due to four commonly isolated, saccharolytic organisms (Alcaligenes xylosoxidans subsp. xylosoxidans, Pseudomonas putida, Pseudomonas fluorescens, and Xanthomonas maltophilia), while 7 of 25 (28%) of misidentifications in the RFP system were due to P. fluorescens. Of note, the RFP system identified non-glucose-fermenting gram-negative bacilli within 2 h of panel inoculation, allowing additional conventional biochemical tests to be set up the same day on low-probability isolates, whereas only 13.5% of the DCPs could be read at 18 h, with the remainder requiring 42 h of incubation before reading. When organism identifications were recalculated with the updated RFP data base and revised software, only 8.1% of all 310 isolates were misidentified at greater than or equal to 85% probability while 77.1% of the isolates were now correctly reported at this same high probability.

Bacteriological Techniques↗

Evaluation of RapID onE system for identification of 379 strains in the family Enterobacteriaceae and oxidase-negative, gram-negative nonfermenters.

The ability of the RapID onE system (Innovative Diagnostic Systems, Inc., Norcross, Ga.) to identify 364 strains in the family Enterobacteriaceae and 15 oxidase-negative, gram-negative, nonfermentative rods was evaluated. Kits were inoculated with no. 2 McFarland standard suspensions, and reactions were interpreted after 4 h of incubation at 35 degrees C. Overall, the method correctly identified (to the species level or to the genus level for salmonellas and non-Shigella sonnei Shigella species) 363 strains (95.8%) without additional tests. For four strains (1.0%), additional tests were required to delineate the correct identification from a range of two or more possibilities; these included one Serratia liquefaciens (Serratia marcescens or Serratia liquefaciens), one Serratia rubidaea (Serratia rubidaea or Serratia odorifera), one Salmonella typhi (Leminorella richardii or Salmonella sp.) and one Yersinia enterocolitica (Yersinia frederiksenii, Yersinia intermedia, or Yersinia enterocolitica). Twelve strains (3.2%) were misidentified or yielded codes with no identification; these comprised one Citrobacter amalonaticus (no identification), three Enterobacter hormaechei (not in the RapID onE database; two Enterobacter amnigenus, one Enterobacter sp.), one Serratia liquefaciens (Enterobacter cloacae), one Serratia rubidaea (no identification), four Serratia fonticola (not in RapID onE database; two Enterobacter aerogenes, one Serratia marcescens, one not identified), one Proteus mirabilis (Proteus penneri), and one Proteus vulgaris (Providencia rustigianii). If the seven strains not included in the database had been excluded, correct identification rates would have risen to 97.6% without additional tests and 98.7% with additional tests, with misidentification rates dropping to 1.3%. The RapID onE system is easy to set up and the results are easy to read, and the system provides an accurate, nonautomated commercially available method for the same-day identification of members of the family Enterobacteriaceae and oxidase-negative, gram-negative nonfermenters.

Bacteriological Techniques↗

Oligonucleotide (GTG)5 as a marker for Mycobacterium tuberculosis strain identification.

Culture of Mycobacterium tuberculosis provides no information on the identity of a strain or the distribution of such a strain in the community. Strain identification of M. tuberculosis can help to address important epidemiological questions, e.g., the origin of an infection in a patient's household or community, whether reactivation of infection is endogenous or exogenous in origin, and the spread and early detection of organisms with acquired antibiotic resistance. To research this problem, strain identification must be reliable and accurate. Although genetic identification techniques already exist, it is valuable to have genetic identification techniques based on a number of genetic markers to improve the accurate identification of M. tuberculosis strains. We show that oligonucleotide (GTG)5 can be successfully applied to the identification of M. tuberculosis strains. This technique may be particularly useful in cases in which M. tuberculosis strains have few or no insertion elements (e.g., IS6110) or in identifying other strains of mycobacteria when informative probes are lacking.

Adult↗

Clinical impact of rapid in vitro susceptibility testing and bacterial identification.

During the past decade, a variety of instrument-assisted bacterial identification and antimicrobial susceptibility test systems have been developed which permit provision of test results in a matter of hours rather than days, as has been the case with traditional overnight procedures. These newer rapid techniques are much more expensive than older methods. It has been presumed but not proven that the clinical benefits of rapid testing to patients with infection offset the added cost. The intent of this study was to objectively define the clinical impact of rapid bacterial identification and antimicrobial susceptibility testing. A 1-year study was performed in which infected, hospitalized patients in a tertiary-care, teaching, medical center were randomly assigned to one of two groups: patients for whom identification and susceptibility testing was performed by using a semi-automated, rapid, same-day procedure and those for whom testing was accomplished by using traditional overnight techniques. The two groups were compared with respect to numerous demographic descriptors, and then patients were monitored prospectively through the end of their hospitalization with the aim of determining whether there existed objectively defineable differences in management and outcome between the two groups. The mean lengths of time to provision of susceptibility and identification test results in the rapid test group were 11.3 and 9.6 h, respectively. In the overnight test group, these values were 19.6 and 25.9 h, respectively (P < 0.0005). There were 273 evaluable patients in the first group and 300 in the second group. Other than the length of time required to provide susceptibility and identification test results, no significant differences were noted between the two groups with respect to > 100 demographic descriptors. With regard to measures of outcome, the mean lengths of hospitalization were also the same in both groups. Mortality rates were however, lower in the rapid test group (i.e., 8.8% versus 15.3%). Similarly, statistically significantly fewer laboratory studies, imaging procedures, days of intubation, and days in an intensive or intermediate-care area were observed with patients in the rapid test group. Rapid testing was also associated with significantly shortened lengths of elapsed time prior to alterations in antimicrobial therapy. Lastly, patient costs for hospitalization were significantly lower in the rapid test group. The results of this study indicate the rapid same-day bacterial identification and susceptibility testing in the microbiology laboratory can have a major impact on the care and outcome of hospitalized patients with infection.

Adolescent↗

Species-specific and ubiquitous DNA-based assays for rapid identification of Staphylococcus epidermidis.

Staphylococcus epidermidis is an aerobic gram-positive coccus that is now recognized among the coagulase-negative staphylococci as an etiological agent with an important range of pathogenicity in humans. Several diagnostic kits based on biochemical or immunological reactions can efficiently identify Staphylococcus aureus. However, these tests are often unreliable for the identification of coagulase-negative staphylococcal species including S. epidermidis. Since DNA-based assays for the species-specific identification of S. epidermidis remain unavailable, we have developed such tests in order to improve the accuracy and the rapidity of tests for the diagnosis of S. epidermidis infections. On the basis of the results of hybridization assays with clones randomly selected from an S. epidermidis genomic library, we identified a chromosomal DNA fragment which is specific and 100% ubiquitous for the identification of S. epidermidis. This 705-bp fragment was sequenced and used to design PCR amplification primers. PCR assays with the selected primers were also highly specific and ubiquitous for the identification from bacterial cultures of clinical isolates of S. epidermidis from a variety of anatomic sites. While three strains of S. capitis were misidentified as S. epidermidis with the API Staph-Ident system and 2.5% of the S. epidermidis identifications were inconclusive with the MicroScan Autoscan-4 system, the PCR assay was highly specific and allowed for the correct identification of all 79 S. epidermidis strains tested. The PCR assays developed are simple and can be performed in about 1 h. The DNA-based tests provide novel diagnostic tools for improving the diagnosis of S. epidermidis infections.

Bacteriological Techniques↗

Comparison of use of phenotypic and genotypic characteristics for identification of species of the anamorph genus Candida and related teleomorph yeast species.

A total of 49 type and neotype isolates and 32 clinical isolates of the anamorph genus Candida and related teleomorph genera were obtained from different culture collections and clinical laboratories. Isolates were subjected to two phenotypic methods of identification, Vitek yeast biochemical card (YBC) and API ID 32C, both based on carbohydrate assimilation, and one genotypic method, PCR fingerprinting, based on the detection of DNA polymorphisms between minisatellite-specific sequences with the primer M13 (5' GAGGGTGGCGGTTCT 3'). The correct identification of a strain at the Centraalbureau voor Schimmelcultures was used as the gold standard for the identification of an isolate. When the study was restricted to species included in the respective biochemical databases, the Vitek YBC and API ID 32C systems performed adequately with positive identification rates of 87.3 and 76.8%, respectively. When uncommon species were added to the study, several of which are not included in the databases, the identification efficiencies were 76.5 and 77.5%, respectively. By comparison, all isolates were correctly identified by PCR fingerprinting, with 63 reference species profiles in the databank. Sufficient polymorphisms among the total set of banding patterns were observed, with adequate similarity in the major patterns obtained from a given species, to allow each isolate to be assigned unambiguously to a particular species. In addition, variations in minor bands allowed for differentiation to the strain level. PCR fingerprinting was found to be rapid, reproducible, and more cost-effective than either biochemical approach. Our results provide reference laboratories with an improved identification method for yeasts based on genotypic rather than phenotypic markers.

Base Sequence↗

Cost-effective and rapid presumptive identification of gram-negative bacilli in routine urine, pus, and stool cultures: evaluation of the use of CHROMagar orientation medium in conjunction with simple biochemical tests.

The algorithm for a new identification system was designed on the basis of colony color and morphology on CHROMagar Orientation medium in conjunction with simple biochemical tests such as indole (IND), lysine decarboxylase (LDC), and ornithine decarboxylase (ODC) utilization tests with gram-negative bacilli isolated from urine samples as well as pus, stool, and other clinical specimens by the following colony characteristics, biochemical reactions, and serological results: pinkish to red, IND positive (IND(+)), Escherichia coli; metallic blue, IND(+), LDC(+), and ODC negative (ODC(-)), Klebsiella oxytoca; IND(+), LDC(-), and ODC(+), Citrobacter diversus; IND(+) or IND(-), LDC(-), and ODC(-), Citrobacter freundii; IND(-), LDC(+), and ODC(+), Enterobacter aerogenes; IND(-), LDC(-), and ODC(+), Enterobacter cloacae; IND(-), LDC(+), and ODC(-), Klebsiella pneumoniae; diffuse brown and IND(+), Morganella morganii; IND(-), Proteus mirabilis; aqua blue, Serratia marcescens; bluish green and IND(+), Proteus vulgaris; transparent yellow-green, serology positive, Pseudomonas aeruginosa; clear and serology positive, Salmonella sp.; other colors and reactions, the organism was identified by the full identification methods. The accuracy and cost-effectiveness of this new system were prospectively evaluated. During an 8-month period, a total of 345 specimens yielded one or more gram-negative bacilli. A total of 472 gram-negative bacillus isolates were detected on CHROMagar Orientation medium. For 466 of the isolates (98.7%), no discrepancies in the results were obtained on the basis of the identification algorithm. The cost of identification of gram-negative bacilli during this period was reduced by about 70%. The results of this trial for the differentiation of the most commonly encountered gram-negative pathogens in clinical specimens with the new algorithm were favourable in that it permitted reliable detection and presumptive identification. In addition, this rapid identification system not only significantly reduced costs but it also improved the daily work flow within the clinical microbiology laboratory.

Agar↗

Application of the Sherlock Mycobacteria Identification System using high-performance liquid chromatography in a clinical laboratory.

There is a growing need for a more accurate, rapid, and cost-effective alternative to conventional tests for identification of clinical isolates of Mycobacterium species. Therefore, the ability of the Sherlock Mycobacteria Identification System (SMIS; MIDI, Inc.) using computerized software and a Hewlett-Packard series 1100 high-performance liquid chromatograph to identify mycobacteria was compared to identification using phenotypic characteristics, biochemical tests, probes (Gen-Probe, Inc.), gas-liquid chromatography, and, when necessary, PCR-restriction enzyme analysis of the 65-kDa heat shock protein gene and 16S rRNA gene sequencing. Culture, harvesting, saponification, extraction, derivatization, and chromatography were performed following MIDI's instructions. Of 370 isolates and stock cultures tested, 327 (88%) were given species names by the SMIS. SMIS software correctly identified 279 of the isolates (75% of the total number of isolates and 85% of the named isolates). The overall predictive value of accuracy (correct calls divided by total calls of a species) for SMIS species identification was 85%, ranging from only 27% (3 of 11) for M. asiaticum to 100% for species or groups including M. malmoense (8 of 8), M. nonchromogenicum (11 of 11), and the M. chelonae-abscessus complex (21 of 21). By determining relative peak height ratios (RPHRs) and relative retention times (RRTs) of selected mycolic acid peaks, as well as phenotypic properties, all 48 SMIS-misidentified isolates and 39 (91%) of the 43 unidentified isolates could be correctly identified. Material and labor costs per isolate were $10.94 for SMIS, $26.58 for probes, and $42.31 for biochemical identification. The SMIS, combined with knowledge of RPHRs, RRTs, and phenotypic characteristics, offers a rapid, reasonably accurate, cost-effective alternative to more traditional methods of mycobacterial species identification.

Bacterial Typing Techniques↗

Comparative evaluation of the BD Phoenix and VITEK 2 automated instruments for identification of isolates of the Burkholderia cepacia complex.

We evaluated two new automated identification systems, the BD Phoenix (Becton Dickinson) and the VITEK 2 (bioMérieux), for identification of isolates of the Burkholderia cepacia complex (BCC). The test sample included 42 isolates of the highly virulent and epidemic genomovar III, 45 isolates of B. multivorans, and 47 isolates of other members of the BCC. Rates of correct identification by the BD Phoenix and VITEK 2 were similar when all BCC isolates were considered (50 and 53%, respectively) but differed markedly for genomovar III (71 and 38%; P < 0.01) and for B. multivorans (58 and 89%; P < 0.001). For the BD Phoenix as well as the VITEK 2, taking all 134 isolates of the BCC together, rates of correct identification of clinical isolates (56 and 55%, respectively; n = 85) were higher than those of environmental isolates (21 and 39%, respectively; n = 28). Clinical isolates of genomovar III (n = 27) showed correct identification rates of 81% (BD Phoenix) and 48% (VITEK 2) (P < 0.01). Rates of misidentification for BD Phoenix and VITEK 2 were 9 and 17% for genomovar III, 22 and 7% for B. multivorans, and 36 and 13% for the other BCC members (P < 0.01), respectively. More than half of the isolates misidentified by each instrument were identified as Ralstonia pickettii, Ralstonia paucula (CDC IV C-2 group), Alcaligenes faecalis, Achromobacter spp., or, for the VITEK 2, "various nonfermenters." This study reemphasizes that confirmatory identification of BCC, preferably by molecular methods, is highly recommended.

Automation↗

Identification of catalase-negative, non-beta-hemolytic, gram-positive cocci isolated from milk samples.

This study was undertaken in an effort to improve the identification scheme of catalase-negative, non-beta-hemolytic, gram-positive cocci isolated from milk samples obtained from cows. First, the sensitivity and specificity of the identification procedure currently in use in our laboratory were compared to the results obtained with API 20 STREP strips which were set as the gold standard. Second, a number of other identification tests, which could contribute to increase the sensitivity and specificity of the identification procedure of these microorganisms, were evaluated and selected. The data have shown that there is a necessity to review the identification procedure. Some modifications are suggested to laboratories doing milk sample analyses. A standardized procedure, using the CAMP test, esculin and sodium hippurate hydrolysis, the presence of the enzymes pyrolidonyl arylaminase and leucine aminopeptidase, and acid production from 1% inulin and raffinose broth, would not only improve the results of the identification process of gram-positive cocci isolated from milk samples but also ensure greater uniformity of the epidemiological data.

Animals↗

Application of an rRNA probe matrix for rapid identification of bacteria and fungi from routine blood cultures.

One of the most important functions of the clinical microbiology laboratory is the identification of the etiology of sepsis. For this study, aliquots from 405 positive blood cultures were tested against a unique array of DNA probes directed against rRNA subsequences of bacteria and fungi for identification. Another 280 samples that were negative after 5 days of incubation were also tested. Blood culture bottles were incubated in a BacT/Alert3D instrument. For the rRNA assay, a 0.4-ml aliquot was removed, and the cells were pelleted by centrifugation. The pellet was washed and frozen at -70 degrees C. Analysis of the pellet involved a lysis step and then the addition of samples to the reaction wells containing the probes in a microtiter plate format. Analysis was performed by using a hybridization protection assay. Results were taken through a series of deductive steps to obtain species, or in some cases genus, identification. Batch sample preparation required approximately 15 min, and sample analysis required another 60 min. Probe results were compared to conventional biochemical identifications. The probe test was negative for the 280 samples that were negative by the BacT/Alert 3D system and for another 21 samples that were false positive (the instrument signaled, but there was no growth). Microorganisms from the remaining 384 signal-positive samples included 60 Enterobacteriaceae, 10 Pseudomonas aeruginosa, 10 other gram-negative bacteria, 40 Staphylococcus aureus, 152 coagulase-negative staphylococci, 28 streptococci, 22 enterococci, 21 other gram-positive bacteria, 8 anaerobes, and 16 yeast organisms. Seventeen cultures were polymicrobial, and one was gram positive and culture negative. Discordance between probe and conventional identification results was noted for only 12 (1.75%) samples. This novel rapid molecular approach to the identification of bacteria and yeast in blood cultures was highly sensitive (100%) and specific (96%).

Automation↗