The identification rates and identification levels were different for each Streptococcus species. Our data indicate that the Rapid Strep system will identify nearly ...
Vol. 20, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1984, p. 894-898 0095-1137/84/110894-05$02.00/0 Copyright © 1984, American Society for Microbiology
Evaluation of the Rapid Strep System for the Identification of Clinical Isolates of Streptococcus Species RICHARD R. FACKLAM,l* D. L. RHODEN,-
AND
P. B. SMITH
Division of Bacterial Diseases1 and Hospital Infections Pr-ogram,2 Center fro Inlfec tiou.s Disetases, Ceniter-s for Disease Control, Atlacnta, Georgia 30333 Received 25 June 1984/Accepted 10 August 1984
A total of 247 strains of streptococci isolated from humans
were
tested for identification in the Rapid Strep
system. The identification rates and identification levels were different for each Streptococcus species. Our data indicate that the Rapid Strep system will identify nearly all the beta-hemolytic Streptococcus species if
serological procedures are used in conjunction with the rapid physiological procedures. Of the group D streptococci, 98% of the enterococci and 95% of the non-enterococci were correctly identified. Of the commonly occurring viridans species, 85% were correctly identified, but only 10% of the less frequently occurring viridans species were identified. A total of 90% of the Streptococcus pneumoniae and 60% of the Aerococcus strains were correctly identified.
terminology conforms to the list of accepted species names approved by the International Congress for Microbiology (10). In this paper we describe our evaluation of a commercial product, the Rapid Strep system (DMS Laboratories, Inc., Flemington, N.J.), designed to identify all species of streptococci within 24 h. (This study was presented in part at the 84th Annual Meeting of the American Society for Microbiology [R. R. Facklam, D. L. Rhoden, A. 0. Esaias, and P. B. Smith, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, C127, p. 257].) MATERIALS AND METHODS Streptococcus strains. All strains tested were clinical isolates from humans and were selected from the Streptococcus Reference Laboratory culture collections at the Centers for Disease Control, Atlanta, Ga. All strains were gram-positive cocci arranged in chains or clusters. All streptococci were catalase negative when tested for effervescence of hydrogen peroxide. All Aerococclus strains were cytochrome negative when tested for cytochromes by the benzidene test. Strains were reidentified by the serological and physiological procedures described in the Manual of Clinical Microbiology (3). Rapid Strep evaluation. Rapid Strep strips and all required reagents were provided by DMS Laboratories, Inc. The majority of the strains were inoculated onto Columbia sheep blood agar plates and incubated anaerobically at 35°C for 18 to 20 h. Of the 247 strains, 75 were inoculated onto Trypticase soy (BBL Microbiology Systems, Cockeysville, Md.) sheep blood agar plates and incubated in a candle extinction jar at 35°C for 18 to 20 h. The manufacturer's instructions call for the use of Columbia sheep blood agar and an anaerobic incubation atmosphere, but other investigators have successfully used other media and incubation atmospheres for the preparation of an inoculum (9). The individuals performing the Rapid Strep test had no knowledge of the identity of the strains other than the hemolytic reaction. Strips were inoculated and read in accordance with the manufacturer's instructions. The Rapid Strep system consists of a strip containing 20 dehydrated substances for determining physiological characteristics. The cupules containing the dehydrated substances were inoculated with a bacterial suspension (equal to or
The procedures used for the conventional identification and differentiation of streptococci are complex and timeconsuming. Serological methods are generally used to identify the different beta-hemolytic streptococci. Although these procedures do not differentiate the beta-hemolytic strains into true taxonomic species, they do enable clinical microbiologists to convey useful information to physicians for managing patients. The development of slide agglutination tests for the rapid identification of beta-hemolytic streptococci has shortened the time required for identification from 3 days (conventional extraction and capillary precipitin testing) to 1 day (slide agglutination testing from primary plates). Direct antigen testing of throat swabs may shorten the time required for group A Streptococcus identification even further (5, 11). Thus, it is not likely that any rapid system based on physiological characteristics will compare with serological techniques in terms of rapidity. However, as more than one species may possess a single group antigen (Streptococcus equi, S. equisimilis, and S. zooepidemicus all have the group C antigen), any system that can accurately differentiate the species within a serological group will be of value. A variety of physiological tests are used to differentiate the non-beta-hemolytic streptococci. Some of these tests are highly specialized and are used only to identify certain Streptococcus species (2, 3). These conventional physiological tests require from 3 to 7 days of incubation, so final identification is delayed to the point that it has very little impact on patient management. Thus, any rapid system that would yield accurate results and be more convenient and time saving would be of significant value to microbiologists and physicians. We have used the terminology described in a recent editorial to designate the Streptococcus milleri group of streptococci (4). S. anginosus will be used to identify the beta-hemolytic minute-colony forms of group A and C streptococci as well as the group F and beta-hemolytic Streptococcus strains without group antigens. S. constellatus will be used to identify strains formerly termed S. anginosus-constellatus, and S. intermedius will be used to identify strains formally termed S. MG-intermedius. This *
Corresponding author. 894
VOL. 20, 1984
STREP SYSTEM FOR IDENTIFICATION OF STREPTOCOCCI
895
TABLE 1. Levels of identification of beta-hemolytic streptococci by the Rapid Strep system No. of strains identified at indicated level within 4 h (by 24 h) Group
A B C
G C A C F None a b
Species (no. of strains)
S. pyogenes (12) S. agalactiae (10) S. equisimilis" (10) Undesignated (10) S. zooepidemicus (10) S. anginosusb (4) S. anginosusb (4) S. anginosusb (8) S. anginosus" (7)
Excellent
Good
Acceptable
1 9 (1) 0 0 6 (4) 4 2 (1) 7 7
6 0 1 6 0 0 0 (1) 1 0
0 0 1 2 0 0 0 0 0
L
ro Licimntow discriminationEro
5 0 5 (3) 2 0 0 0
0 0
0 0 0 0 0 0
0 0 0
Identified by the Rapid Strep system as group C S. equisimilis or group G streptococci. Identified by the Rapid Strep system as S. milleri (see text).
greater than a McFarland no. 4 turbidity standard) prepared from the agar plates described above. Reactions were read at 4 h and after overnight incubation (1). Seven-digit profile numbers were obtained by adding the numerical values for the test results in the strip (see manufacturer's identification code book for details.) The identification and level of identification for each strain were obtained by matching the profile number to the profile identification number provided in the manufacturer's code book. The identification of a strain is based on the similarity between its profile and the taxa that constitute the computerstored data base. The level of identification is based on a combination of the different reaction frequencies for each taxon with each test. The computer-generated number is converted to a percentage, which is known as the percentage identification or level of identification. Profile numbers that were not listed in the code book were sent with conventional identification to the manufacturer for analysis. The manufacturer then provided the final identification. The levels of identification that we used (see Tables 1, 2, and 3) corresponded roughly to the manufacturer's suggestions. We combined the excellent and very good categories into one category, excellent. The percentage identification of our excellent was equal to or greater than 99.0%. The good and acceptable categories were those suggested by the manufacturer and corresponded to 90.0 to 98.9% and 80.0 to 89.9% identification, respectively. We combined two other levels of identification, low discrimination and species identification, into one category, low discrimination. Both categories, as suggested by the manufacturer, had identification levels below 80.0% and required additional testing to confirm the identity of the test strain. The tests used to discriminate between the two or three possible identifications are listed in the code book. Our last level of identifica-
tion, error, included strains that were identified at the acceptable level or above but whose identity did not correspond to the conventional identification. The error level also included strains that generated profile numbers not included in the code book and for which the manufacturer was unable to produce an identification because of an inadequate data base. RESULTS The results of testing 75 strains of beta-hemolytic streptococci are shown in Table 1. Serological testing was suggested for confirmatory identification of all strains of group A, B, C (S. equisimilis), and G streptococci, r'egardless of the level of identification. Discrimination between group C S. equisimilis and group G streptococci is not possible by the physiological tests in the Rapid Strep system. All 10 group C S. zooepidemicus strains were identified at the excellent level. Serological confirmation was not suggested for these strains. The last four lines of Table 1 include four serological entities of one Streptococcus species, S. anginosus. The Rapid Strep system identified these strains as S. milleri. The differences in nomenclature between the Centers for Disease Control and the Rapid Strep system will be discussed later. All the group A, C, and F strains and nongroupable strains of S. anginosus were identified at the excellent and good levels as S. milleri type 1, with the exception of one strain of group C S. anginosus. It was identified as S. milleri type 2. Serological confirmation was not suggested for these strains. No identification errors were observed for the beta-hemolytic strains (Table 1), and 63 of the 75 strains (84%) were identified within 4 h; the remainder of the strains were identified after overnight incubation. The profile numbers of two strains, both group C S. equisimilis, were not included in the code book. The manufacturer's analysis of these two
TABLE 2. Levels of identification of non-beta-hemolytic strains of group B and D streptococci and aerococci by the Rapid Strep system Group
Species (no. of strains) Excellent
B D D D D D D None
S. agalactiae (4) S. faecalis (10) S. faecium (11) S. durans (3) S. avium (10) S. bovis 1(10) S. bovis II (12) Aerococcus spp. (10)
2 (2) 0 4 0 0 (9) 8 (2) 9 6
No. of strains identified at indicated level within 4 h (by 24 h) Low
Good
Acceptable
0 10 3 0 (2) 0 (1) 0 2 0
0 0 0 0 0 0 0 0
discrimination 0 0
0 (3) 0 (1) 0 0 0 0
Error
0 0 0 (1) 0 0 0 0 (1) 0 (4)
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J. CLIN. MICROBIOL.
FACKLAM, RHODEN, AND SMITH
TABLE 3. Levels of identification of viridans streptococci and pneumococci by the Rapid Strep system Species (no. of strains)
S. S. S. S. S. S. S. S. S. S. S.
acidominimus (5) constellatus' (10) intermedius' (14) mitis (10) morbillorum (10) mutans (10) salivarius (10) sanguis I (10) sanguis II (10) uberis (7) pneumoniae (10)
No. of strains identified at indicated level within 4 h (by 24 h) Good
Excellent
0 0 0 0 0 0 0 1 0
0 5 5 0 0 0 0 1 0
(1)
(1) (10)
(10) (3) (4) (2) (1) (4) (2) X0 3 1
Low
Acceptable
0 1 0 0 0 0 0 2 0 0 1
0 0 0 0 0 0 0 0 0 (2) 0 0
Error
(1) (5)
(1) (3) (2)
(4)
4 3 3 0 10 0 2 0 0 6 1
Identified by the Rapid Strep system as S. milleri (see text).
numbers resulted in a low-discrimination level of identification for both strains. The results of testing 4 strains of nonhemolytic group B S. agalactiae, 56 strains of six different group D Streptococclus species, and 10 Aerococcus strains are shown in Table 2. The levels of identification were the same as described above. Serological testing was suggested for confirmation of the identities of the group B but not of the group D streptococcal species. Of the 70 strains, 60 (86%) were identified at the excellent or good level. Of the 10 Aerococcus strains, 6 were identified at the excellent level, but 4 remained unidentified because the profile numbers that were not included in the code book and analysis of the profile numbers by the manufacturer did not result in identification. A total of 66% of the strains were identified within 4 h and the remainder of the strains (except the errors) were identified after overnight incubation. In addition to the profile numbers of the four unidentified Aerococcus strains, the profile numbers of two S. faecium, one S. avium, and one S. durans strains had to be analyzed by the manufacturer. The analysis of these latter four numbers resulted in a lowdiscrimination, but correct, level of identification for all of the strains.
The results of testing 96 strains of viridans streptococci and 10 strains of pneumococci are shown in Table 3. A total of 10 strains of each of the viridans species were tested with the exception of S. acidominimus and S. uberis, for which only 5 and 7 strains, respectively, were available. Four strains of the mannitol-positive variant of S. intermedius (unidentified urine isolates [9]) were also tested. Of the 106 strains, 55 (52%) were identified at the excellent or good level. Of the 96 viridans strains, 20 were identified at the low-discrimination level, and 29 either were not identified because of unsatisfactory profile pumbers or were erroneously identified. The seven correctly identified strains of S. constellatus were identified as S. milleri type 1 by the Rapid Strep system. Of the eight correctly identified strains of S. intermedius, seven and one were identified as S. milleri types 2 and 3, respectively, by the Rapid Strep system. Of the four mannitol-positive strains of S. intermedius, two and one were identified as S. milleri types 3 and 2, respectively, by the Rapid Strep system. None of the 10 strains of S. morkillorum were identified by the Rapid Strep system because no data base exists for this species. Confirmation of identities by testing for optochin sensitivity was suggested for all correct identifications of pneumococci.
TABLE 4. Analysis of Rapid Strep system identification errors Conventional
Atypical
identification (no. of strains)
Atypical
S. faecium S. bovis 11 Aerococcus sp. (2) Aerococcus sp. (2) S. acidominimus (4) S. constellatus S. constellatus S. constellatus S. intermedius
characteristics" Arabinose -
Inulin
+
None
Hippurate
-
None None Bile-esculin +
Arginine Arginine -,
Rapid Strep system Identification
Excellent (98.9) Low discrimination (
