Rapid presumptive identification of gram-negative rods directly from blood cultures by simple enzymatic tests.

Gram-negative rods were presumptively identified directly from blood cultures within 15 min as Escherichia coli, a member of the Klebsiella-Enterobacter group, or oxidase positive. Samples of artificially seeded blood cultures (193 cultures) and patient blood cultures (78 cultures) were filtered into a Dynadepth test card with the Bac-T-Screen instrument (Vitek, Inc., Hazelwood, Mo.). Triton X-100 was then filtered into the test card to lyse the blood cells but not the entrapped bacteria, and either methylumbelliferone-labeled substrates or oxidase reagent was applied to the filter surface. The oxidase test was read within 30 s, and the methylumbelliferone and indole tests were read after a 10-min incubation at room temperature. Positive beta-galactosidase, beta-glucuronidase, and indole test results predicted the identification of E. coli with a 96 to 100% sensitivity and a 99 to 100% specificity. Positive beta-xylosidase and beta-galactosidase test results and negative oxidase and beta-glucuronidase test results were 85 to 93% sensitive and 100% specific for a Klebsiella-Enterobacter organism. A positive oxidase test result and negative beta-glucuronidase, beta-xylosidase, and indole test results were highly predictive of Pseudomonas aeruginosa (sensitivity, 100%; specificity, 99%). The procedures described are rapid and simple and provide a direct presumptive identification of the gram-negative rods most commonly found in blood cultures.     

Rapid presumptive identification of anaerobes in blood cultures by gas-liquid chromatography.

Production of volatile and nonvolatile metabolic acids in blood culture broths by aerobic, facultative anaerobic, and obligate anaerobic bacteria was analyzed by gas-liquid chromatography. Anaerobic blood culture isolates were presumptively identified by the qualitative analysis of volatile fatty acids. Isolates, with a characteristic Gram stain reaction and cellular morphology, were identified by the following acid patterns: Bacteriodes fragilis group with acetic and propionic acids; Fusobacterium with acetic, butyric, and usually propionic acids; Veillonella with acetic and propionic acids; gram-positive cocci with acetic and butyric acids; and Clostridium with acetic and butyric acids.     

Rapid method for identification of group B streptococci in neonatal blood cultures.

A rapid technique used for the identification of Streptococcus agalactiae, Lancefield group B, from the blood cultures of two neonatal infants is reported. The method utilized the Phadebact Streptococcus Test System (Pharmacia Diagnostics, Piscataway, N.J.) and the supernatant from 13- and 14-h blood cultures. Additional studies with simulated neonatal blood cultures revealed that this method was reproducible. Additional studies also revealed that some non-specific agglutination did occur, which could not be eliminated with dithiothreitol, but was visibly reduced by treatment with soluble staphylococcal protein A.     

Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR

A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.     

Evaluation of Phadebact and Streptex Kits for rapid grouping of streptococci directly from blood cultures.

The Phadebact Streptococcus Test and the Streptex Test kits were evaluated for grouping streptococci directly from blood cultures. Pellets of bacteria obtained from centrifuged samples of positive blood cultures were inoculated into Todd-Hewitt broth for 2- and 4-h Phadebact tests and into pronase for Streptex tests. Hemolysis was determined after pipetting a portion of each pellet into cuts made in blood agar plates incubated anaerobically for 2 to 6 h. Serological groups were also determined from colonies of the 137 strains of streptococci used in the study by the Lancefield precipitin method. Of the 126 strains tested by the 4-h Phadebact method, 120 (95.2%) agreed with Lancefield groupings, and 133 (97.1%) of the 137 strains tested by Streptex were in agreement. In contrast, only 31 of 55 strains (56.4%) were correctly identified by the 2-h Phadebact method. Misidentifications were related to multiple agglutinations and weak agglutinations in homologous antisera. Group A isolates were most frequently misidentified by all of the test methods. Hemolysis was determined within 4 h for 92.7% of the isolates and within 6 h for the remaining strains. Although the 4-h Phadebact procedure and the Streptex procedure were comparable in overall accuracy, cost, and technologist time, Streptex was the method of choice for direct groups. Results were available within 75 min for the Streptex procedure compared with 4 h for the Phadebact method. Because few cross-reactions occurred, agglutination responses were clearer and easier to interpret. Results from 2-h Phadebact tests were not satisfactory, and this method is not recommended.     

Rapid identification of gram-negative bacilli from blood cultures

Blood-culture broths showing macroscopic or radiometric evidence of growth of gram-negative bacilli were examined by a rapid automated bacterial identification system. A differential centrifugation technique was developed to prepare suitable inocula. The identification results obtained were confirmed by the API 20E method, with single colonies of the strains isolated 24 h later. Of 90 organisms tested, seven did not give the same identification by the two systems. With the rapid automated technique a presumptive identification of gram-negative bacilli can be made 24 h earlier than by more conventional methods.     

Rapid immunodiagnosis of streptococci and enterococci in blood cultures

We evaluated a procedure for rapid identification of blood culture isolates of pneumococci, enterococci and beta-haemolytic streptococci groups A, B, C, and G. Immunological tests were applied directly to blood culture medium and included a quellung reaction for pneumococci, and latex agglutination tests (LAT) for pneumococcal antigen and Lancefield antigens A, B, C, D and G. During a one-year trial period with approximately 12,000 blood culture sets (BacT/Alert, Organon-Teknika), 208 sets showed pure growth of gram-positive cocci in pairs or chains by direct microscopy. Overall, a correct diagnosis was noted for 103 (63.2%, 95% CI: 55.3-70.6%) of 163 sets yielding pneumococci, beta-haemolytic streptococci, or enterococci. A procedure excluding Lancefield antigens only A, B, and D was implemented during a one-year follow-up period. With this simplified procedure positive and negative predictive values, respectively, were 1.00 and 0.87 for pneumococci, 1.00 and 0.94 for beta-haemolytic streptococci group A, 0.60 and 1.00 for beta-haemolytic streptococci group B, and 0.91 and 0.88 for enterococci. We conclude that rapid identification of gram-positive cocci is feasible and may improve the information given to clinicians at the first notification of positive blood cultures.     

Rapid biochemical tests for the identification of groups A, B, C, F, and G streptococci from throat cultures.

A test employing three fluorogenic 4-methylumbelliferyl substrates and the lectin of Dolichos biflorus was developed for the identification of beta-hemolytic streptococcal colonies associated with throat cultures. This non-serological method is unique in that it permits the accurate identification of groups C, F, and G streptococci, as well as groups A and B streptococci. The method is rapid, simple, and specific and appears to be a useful means to identify groups A, B, C, F, and G streptococci.     

Rapid identification of Staphylococcus aureus and Streptococcus pneumoniae from blood cultures.

Simultaneous application of the lysostaphin sensitivity test for identification of Staphylococcus aureus and the deoxycholate test for the identification of Streptococcus pneumoniae was evaluated for reliability in rapid identification (1 h) of these organisms from blood cultures by using BACTEC 6B and 7C bottles. The procedure was applied to 127 cultures, 74 lysostaphin tests and 53 deoxycholate tests. Lysostaphin-tested organisms included 23 S. aureus, 40 Staphylococcus epidermidis, 1 Listeria sp., 1 Peptococcus sp., 2 Micrococcus sp., 2 diphtheroids, and 5 mixed cultures. Deoxycholate-tested organisms included 14 S. pneumoniae, 32 Streptococcus spp. (not S. pneumoniae), 1 Listeria sp., 1 Peptococcus sp., 2 diphtheroids, and 3 mixed cultures. Analysis of the data revealed positive and negative predictive values of 95.8 and 94%, respectively, for the lysostaphin test and 100 and 97.4%, respectively, for the deoxycholate test. The combined tests provide rapid reliable identification or elimination of S. aureus and S. pneumoniae in blood cultures.     

Rapid presumptive identification of Cryptococcus neoformans.

Carbohydrate-containing extracts were prepared from mature yeast colonies grown on Sabouraud dextrose agar by mixing a 0.001-ml loopful of yeast cells for 30 s in phenolized saline and removing the cells by centrifugation. Extracts were prepared from 54 Cryptococcus neoformans isolates, 29 isolates of other Cryptococcus species, 16 isolates of Candida species, 2 Rhodotorula, 2 Torulopsis, and 1 Saccharomyces species. Initially the carbohydrate content of each extract was estimated (Molisch method) and adjusted to 1, 5, and 10 microgram/ml. Twofold dilutions of each extract were tested for reactivity with the cryptococcal latex agglutination reagent of Bloomfield et al. (N. Bloomfield, M.A. Gordon, and D.F. Elmendorf, Jr., Proc. Soc. Exp. Biol. Med. 114:64-67, 1963). All 54 C. neoformans extracts gave strong agglutinations (3+ to 4+) in dilutions of 1:4 or greater. None of the other yeasts produced any agglutination, except for 1 of 15 C. laurentii isolates, which showed a 1+ reaction that disappeared at a dilution of 1:4 and above. Subsequent testing established that a single extract made from 0.001 ml of yeast cells in 6 ml of phenolized saline contained less than 5 microgram of carbohydrate per ml, was suitable for a single rapid screening dilution, and eliminated any cross-reaction from the C. laurentii isolates. In our hands this method has provided a reliable differentiation of C. neoformans from other unknown yeast colonies in less than 20 min exclusive of a Molisch determination.     

Rapid detection of group B streptococci directly from vaginal swabs.

Duplicate vaginal swabs were obtained from patients who attended obstetric or gynecologic clinics affiliated with the Magee Womens Hospital in Pittsburgh. One swab was cultured semiquantitatively on 5% sheep blood agar to detect group B streptococci (GBS). The other swab was subjected to a rapid method (25 min) for antigen detection and micronitrous acid exposure to extract the GBS antigen, followed by latex particle agglutination. A total of 464 swabs were evaluated by direct plating. Fifty-two swabs (11.2%) were found to contain GBS. Overall, the rapid method detected 21 of 52, or 40.4%, positive specimens. The sensitivity of the rapid method for identifying the most heavily colonized samples was 85.7%. This method can be used to identify maternity patients who are heavily colonized with GBS and are at high risk of delivering septic infants.     

CAMP-disk test for presumptive identification of group B streptococci.

Standaridizing test results for rheumatoid factor by comparing results obtained for an unknown with results obtained for a serum reference preparation decreased variance between laboratories, as measured in the Center for Disease Control proficiency testing program, by 77%. The amount of improvement was also estimated by the type of test and by the manufacturer's product. Standardization resulted in an increase in the number of reported results that were within a twofold dilution of the median value. The percentage increased from 50.3 to 93.7% for the slide tests and from 78.1 to 91.2% fro the tube tests. Decrease in variance by manufacturer's product ranged from 94 to 27%. The study demonstrated that adopting a reference serum standard could substantially improve the comparability of rheumatoid factor test results and that proficiency testing programs can be used to estimate improvement which could be expected as a result of standardization.     

Rapid presumptive identification of Cryptococcus neoformans by staphylococcal coagglutination.

A coagglutination reagent was prepared by sensitizing the Cowan I strain of Staphylococcus aureus with rabbit immune globulin directed against Cryptococcus neofromans A15 and absorbed with C. laurentii. This reagent was evaluated for its usefulness in differentiating C. neoformans from other yeast colonies rapidly. Antigen-containing extracts were prepared form Sabouraud dextrose agar cultures of 48 C. neoformans, 33 other Cryptococcus species, 21 Candida, 4 Torulopsis, 3 Saccharomyces, and 2 Rhodotorula strains. This was done by suspending a 0.001-ml loopful of colony growth in 0.5 ml of phenolized saline, mixing for 30 s, and then centrifuging. Equal volumes (50 microliters) of coagglutination reagent and yeast extract were mixed within marked circles on a glass slide and then mechanically rotated at 180 rpm for 8 min. Forty-five of the 48 strains of C. neoformans produced strong (3+ to 4+) agglutination, and 3 strains of serotype C produced weak (1+ to 2+) agglutination with the reagent. Other Cryptococcus species which reacted positively were 4 C. albidus subsp. diffluens, 7 C. albidus subsp. albidus, and 2 C. terreus strains; however, false-positive errors in identification were circumvented by performing a supplemental rapid test for nitrate utilization which differentiated these yeasts from C. neoformans. None of the other yeasts tested (including 14 C. laurentii, 2 C. luteolus, and 2 C. uniguttulatus strains) produced any degree of agglutination with the reagent. A commercial cryptococcal latex agglutination reagent (Crypto-Test, Microbiological Associates, Walkersville, Md.) proved less reliable for identifying C. neoformans yeast colonies because of cross-reactions which occurred with all other species of Cryptococcus tested.     

Rapid identification and antimicrobial susceptibility testing of gram-negative bacilli from blood cultures by the AutoMicrobic system.

A procedure was developed which allows direct identification and antimicrobial susceptibility testing of fermentative and nonfermentative gram-negative bacilli from positive blood cultures. A 10-ml sample was removed from turbid blood culture bottles, and the bacteria were washed and concentrated by centrifugation. The bacterial pellet was used to inoculate an Enterobacteriaceae Plus Identification Card and a Gram-Negative General Susceptibility Card of the AutoMicrobic system. Results with these cards were compared with results obtained with standard technique for 196 blood cultures seeded with recent clinical isolates. Identification of most cultures was available in 8 h, whereas the antimicrobial susceptibility results were available in an average of 4.7 h for all organisms. Direct identification was correct for 95% of the cultures, whereas the antimicrobial susceptibility data had an average agreement of 87% with 3.8% very major and 1.4% major errors. In using this procedure it was possible to provide accurate preliminary identification and results of antimicrobial susceptibility tests for gram-negative bacilli on the same day that a blood culture was determined to be positive.     

Rapid identification of group B streptococci by counterimmunoelectrophoresis.

Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this techinque is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains.     

Rapid identification of Staphylococcus aureus in blood cultures by thermonuclease testing.

The detection of thermonuclease activity in 86 blood culture samples containing gram-positive cocci showed 100% correlation with the subsequent identification of the isolate as Staphylococcus aureus by the coagulase test. No positive thermonuclease results were found with 66 samples containing coagulase-negative staphylococci and 56 samples containing other gram-positive organisms. The thermonuclease test provides a rapid, reliable method to identify S. aureus in blood cultures.     

Direct identification of gram-positive cocci from routine blood cultures by using AccuProbe tests

Rapid and reliable identification of bacteria directly from blood cultures is important in clinical practice to guide appropriate antibiotic therapy. In this study, the performance of the AccuProbe (Gen-Probe, Inc., San Diego, Calif.) in direct identification of Staphylococcus aureus, Streptococcus pneumoniae, enterococci, and group A and B streptococci from positive blood culture bottles was evaluated by using 6-year routine clinical laboratory blood culture material from Paijat-Hame Central Hospital, Lahti, Finland. With the enterococcal and group A and B streptococcal probes, the diagnostic performance of the test was excellent at a cutoff value of 50,000 relative light units (RLU) as recommended by the manufacturer. However, with the S. aureus probe, although the specificity was very high (99.8%), the sensitivity was low (72.4%). To improve the clinical usability of the direct AccuProbe identification, optimal cutoff values for the individual AccuProbe tests were defined by using receiver-operating characteristic analysis. Consequently, cutoff values for S. aureus and S. pneumoniae tests were adjusted to 30,000 RLU and for enterococci and to 55,000 RLU for group A and B streptococci. With these adjustments, the performance of the AccuProbe tests, especially that for S. aureus, was significantly improved.