DIAGN MICROBIOLINFECTDIS 1992;15:295-300
295
Rapid, Direct Identification of Staphylococcus aureus and Streptococcus pneumoniae from Blood Cultures Using Commercial Immunologic Kits and Modified Conventional Tests Thomas E. Davis, DeAnna D. Fuller, and Ellen C. Aeschleman
To develop safe and rapid methods for identification of Staphylococcus aureus and Streptococcus pneumoniae directly from positive blood culture bottles (BCB) (BACTEC,Johnston Laboratories), several commercial biochemical and immunological tests as well as modified conventional tests were evaluated. Preliminary studies demonstrated that both S. aureus and St. pneumoniae could be identified directly using only a small aliquot (100 tzl) of the blood culture broth obtained via vent without need for centrifugation or other separation steps. A simple tube coagulase exhibited 98% sensitivity and 100%
specificity for 32 S. aureus isolates and 157 blood cultures positive for coagulase-negative staphylococci when read at 2 hr. All systems employed for direct identification of St. pneumoniae exhibited excellent sensitivity and specificity using " aliquots from blood culture broths, but Pneumoslide (BBL Microbiology Systems, Cockeysville, MD) was easiest to perform and interpret. The results of this study show that S. aureus and St. pneumoniae can be identified directly from blood culture broth aliquots using rapid methods that eliminate the need for centrifugation or use of needles and syringes.
INTRODUCTION
tients (Fidalgo et al., 1990). Early identification of staphylococci as coagulase negative may prevent unnecessary antibiotic therapy (Dunagan et al., 1989; Kunin et al., 1990). Bacteremias caused by S. aureus and St. pneumoniae require prompt and appropriate antibiotic therapy. They account for 50% of community-acquired bacteremias in AIDS patients, and rapid diagnosis can optimize therapy (Krumholz et al., 1989). Other Gram-positive cocci, especially enterococci, are often resistant to multiple antibiotics, and rapid identification guides choice of therapy (Sahm et al., 1989; Uttley et al., 1988). The direct identification of blood culture isolates has recently been reviewed by Strand and Shulman (1988) and, in most situations, agreement between rapid and conventional methods has been good. Rapid commercial tests have been applied directly to bacteria from blood cultures (Flejzor and Bokkenheuser, 1984; Hamoudi et al., 1988; Rappaport et al., 1988; Shlaes et al., 1984). Methods for rapid identification
The most common blood culture isolates at this and other institutions, are Gram-positive cocci (Bryan 1989; Rappaport et al., 1988). Among the Gram-positive cocci, coagulase-negative staphylococci, Staphylococcus aureus, and Streptococcus pneumoniae have been most frequent. Coagulase-negative staphylococci are frequently found in blood cultures as contaminants from the skin, but may be pathogenic in some paFrom the Wishard MemorialHospital--IndianaUniversity Medical Center (T.E.D., D.D.F., E.C.A.), Indianapolis, Indiana; and Watauga Hospital Inc. (E.C.A.), Boone, North Carolina, USA. Address reprint requests to Dr. T.E. Davis, Wishard Memorial Hospital, Department of Pathology, 1001 West 10th Street, Indianapolis, IN 46202, USA. Received 28 March 1991; revised and accepted 19 August 1991. © 1992 ElsevierScience Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00
296
of Gram-positive cocci have included Gram stain, coagglutination, and latex agglutination (Agger and Maki, 1978; Rappaport et al., 1988; Severance et al., 1980). We decided to compare both commercial immunologic identification systems and conventional modified laboratory tests for direct and rapid identification of blood culture isolates of S. aureus and St. pneumoniae. We arbitrarily chose 2 hr as the maximum time for completion of these tests.
MATERIALS AND METHODS Processing Positive Blood Cultures Blood cultures collected from patients at Wishard Memorial Hospital, Indianapolis, Indiana, were analyzed from January 1989 to June 1990. Blood (3-5 ml) was collected into BACTEC6A (NR) and 7A (NR) and/or 16 (NR) and 17 (NR) blood culture bottles (BCB) (Johnston Laboratories, Towson, MD). A blood culture bottle (BCB) with a growth index of 30 or greater by the BACTEC method was Gram-stained and if Gram-positive cocci were observed, then the Gram smear was categorized into one of three groups: Staphylococcus-like, Streptococcus-like, or equivocal.
Identification of Isolates Gram-positive cocci isolated from blood cultures were identified by standard laboratory methods (Facklam and Carey, 1985; Kloos and Jorgenson, 1985; Hendrickson, 1985). For presumptive identification of Gram-positive cocci directly from blood culture bottles, several methods were employed. Conventional tests included Gram's stain, slide bile solubility, NaC1Esculin test, slide coagulase, and the tube coagulase. Commercial kits evaluated were Staphaurex, Streptex, and Wellcogen Strep Test (Wellcome Diagnostics, Research Triangle Park, NC), Staphyloslide and Pneumoslide (BBL Microbiology Systems, Cockeysville, MD), and Thermonuclease Agar (Remel Laboratories, Lenexa, KS). A 24-hr tube coagulase test was performed on all microorganisms that tested negative for S. aureus at 2 and 4 hr (Kloos et al., 1985). Tests used for identification of St. pneumoniae included the slide modification of the bile solubility test, Pneumoslide, and Wellcogen Strep Test. Microorganisms described morphologically as streptococci that tested negative for St. pneumoniae were tested with Streptex, Fluorescent Antibody method for Group A Streptococcus, a 4-hr NaCI-Esculin broth, and Identicult A-E (Facklam and Carey, 1985). Tests for both staphylococci and streptococci were performed when the smear showed Gram-positive cocci without a predominance of clumps or chains.
T.E. Davis et al.
Polymicrobial bacteremia was rare and not included in this study. For identification of staphylococci and streptococci, three different sample types were used: (a) one drop (-100 ~I) of a well-mixed blood culture broth aliquot (BCBA) taken directly from the positive blood culture bottle using a blood culture bottle vent/subunit (Difco Laboratories, Detroit, MI); (b) 4-5 bacterial colonies representing colonial growth (CG) from agar plates inoculated from positive blood culture bottles and incubated 24 hr at 35°C; and (c) blood culture pellet (BCP) of bacteria collected from the positive blood culture bottles. The pellets were obtained by removing 10 ml of broth from the blood culture bottle and centrifuging at 150 g for 10 min to sediment out the red blood cells. The supernatant was then centrifuged at 1000 g to concentrate the bacteria. The resulting supernatant was removed, and the pellet was resuspended in 1 ml of sterile saline. For all three sample types, specific manufacturer's instructions were followed for test performance. For immunologic identification of staphylococci and streptococci, one drop of BCP, CG, or BCBA was transferred to a well on a test card (supplied with each reagent), and instructions of the manufacturers were followed. For biochemical identification of staphylococci and streptococci, one drop of BCP, CG, or BCBA was transferred to each biochemical tube or plate, and specific procedural instructions for our laboratory were followed.
RESULTS Direct Testing of Staphylococci from Blood Culture Pellets A total of 72 pellets containing staphylococci were examined by a variety of commercial immunological, biochemical, and rapid modifications of conventional identification methods (Table 1). The tube coagulase identified 100% of blood culture bottles containing S. aureus within 2 hr. The sensitivity was 68% with Thermonuclease Agar. The specificity for all methods ranged from 93% (Thermonuclease Agar) to 100% (tube coagulase).
Testing of Staphylococci from Corresponding Colonial Growth A total of 70 isolates obtained from blood culture pellets were subsequently tested with the same combination of tests performed from colonies on solid media on the following day (Table 1). The sensitivities were 100% for all tests, and the specificity ranged from 93% (Staphaurex) to 100% (Slide coagulase, Thermonuclease Agar, Staphyloslide, and tube coagulase).
297
Identification of Staphylococcus aureus a n d Streptococcus pneumoniae
TABLE 1
Identification of Staphylococcus aureus a n d Coagulase-Negative Staphylococci No. of Correct Identifications a
Specimen
Test System
Coagulase-Negative S. aureus Staphylococci
Sensitivity (%)
Specificity (%)
Negative Predictive Value (%)
Positive Predictive Value (%)
BCPb
Staphaurex Slide coagulase Thermonuclease Staphyloslide Tube coagulasec
9/24 12/24 15/22 8/24 24/24
47/48 46/47 41/44 45/46 47/47
38 50 68 33 100
98 98 93 98 100
76 79 85 74 100
90 92 83 89 100
CGa
Staphaurex Slide coagulase Thermonuclease Staphyloslide Tube coagulase
24/24 4/4 24/24 5/5
43/46 11/11 8/8 46/46 11/11
100 100 100 100 100
93 100 100 100 100
100 100 100 100 100
89 100 100 100 100
Staphaurex Slide coagulase Thermonuclease Staphyloslide Tube coagulasec
4/5 5/5 4/4 4/5 31/32
12/13 11/12 8/9 11/12 157/157
80 100 100 80 97
92 92 89 92 100
92 100 100 92 98
80 83 80 80 100
BCBAb
5/5
aThe first number represents correct identifications by the listed method and the second number represents the total number of isolates tested. bBlood cultures (18) containing staphylococci were tested using BCBA(blood culture broth aliquot) only. BCP, blood culture pellet. CAllbut one of the S. aureus isolates were coagulase positive within 2 hr. dColonial growth.
Direct Testing for Staphylococci Using 100-1~1 Blood Culture Broth Aliquots Isolates (189) were tested with the previously listed m e t h o d s (Table 1). The sensitivity r a n g e d from 80%
TABLE 2
(Staphaurex a n d Staphyloslide), to 97% (tube coagulase), to 100% (Slide coagulase a n d T h e r m o n uclease Agar). The specificity r a n g e d from 89% (Thermonuclease Agar) to 92% (Staphaurex, slide coagulase, Staphyloslide) to 100% (tube coagulase).
Identification of Streptococcus pneumoniae a n d from Blood Culture Pellets No. of Correct Identifications a
Specimen
Test System
Other St. pneumoniae Streptococci
Sensitivity (%)
Specificity (%)
Negative Predictive Value (%)
Positive Predictive Value (%)
BCPb
Pneumoslide Bile solubility Wellcogen strep test
23/23 24/24 20/24
19/19 17/17 13/13
100 100 83
100 100 100
100 100 76
100 100 100
CGC
Pneumoslide Bile solubility Wellcogen strep test
22/22 8/8 8/8
13/13 3/3 3/3
100 100 100
100 100 100
100 100 100
100 100 100
BCBAb
Pneumoslide Bile solubility Wellcogen strep test
32/32 23/23 25/26
40/40 5/5 5/5
100 100 96
100 100 100
100 100 83
100 100 100
aThe first number represents correct identifications by the listed method and the second number represents the total number of isolates tested. bBlood cultures (30) containing streptococci were tested using BCBA(blood culture broth aliquot) only. BCP, blood culture pellet. CColonygrowth.
298
Direct Testing of Streptococcus pneumoniae from Blood Culture Pellets Tests for St. pneumoniae were conducted on 42 BCP by using the slide modification of the bile solubility test, Pneumoslide, and Wellcogen St. pneumoniae latex agglutination (Table 2). The slide bile solubility and Pneumoslide yielded sensitivities of 100%, and the Wellcogen latex agglutination had a sensitivity of 83%. Each method yielded a specificity of 100%.
Testing of Streptococcus pneumoniae Using Corresponding Colonial Growth The above three methods were performed the following day on subsequent colonial growth (Table 2). The sensitivity and specificity of each was 100%.
Direct Testing for Streptococcus pneumoniae Using 100-~1 Blood Culture Broth Aliquots This additional procedure was performed using the slide bile solubility and Pneumoslide tests with both yielding sensitivities and specificities of 100% (Table 2). DISCUSSION Our study shows that rapid conventional methods (tube coagulase and slide bile solubility) can be used to identify S. aureus and St. pneumoniae directly from positive blood cultures. The sensitivity and specificity of these inexpensive tests match or surpass the more expensive commercial immunologic or biochemical kits evaluated simultaneously. These simple methods are accurate w h e n performed directly on blood culture broth aliquots and appear to be at least as reliable as those performed on colonial growth or blood culture pellets. As reported by others (Knight and Shlaes, 1983; Rappaport et al., 1988), the slide bile solubility test for St. pneumoniae is accurate but time consuming, as it requires an additional Gram stain and microscopic interpretation. We did not find false-positive tests with this method as experienced by others (Murray 1979). The slide bile solubility test is reliable for testing CG, BCP, and BCBA. We evaluated the Pneumoslide and Wellcogen Strep Test commercial kits and, as has been the experience of others (Gordon et al., 1987; Rappaport et al., 1988), these tests showed excellent sensitivity and specificity w h e n performed on blood culture broth aliquots. Pneumoslide was equivalent to the slide bile solubility test in accuracy and is easier
T.E. Davis et al.
to perform and interpret by our technologists. In our hands, the Pneumoslide test yielded 100% sensitivity and specificity on all specimens. Hamoudi and Hribar (1988) reported numerous false-positive and false-negative results with Staphaurex when attempting to identify S. aureus in blood culture pellets. In agreement with Rappaport et al. (1988), we found Staphaurex and Staphyloslide to be reliable for testing colonies from solid media, but both lacked sensitivity w h e n testing blood culture pellets. The tube coagulase test exhibited 100% specificity and failed to detect only one S. aureus. Further, the tube coagulase is simple to perform and fits well into the workflow of our blood culture bench. The slide coagulase method also works well, but requires more hands-on time. Although others (Faruki and Murray, 1986; Madison and Baselski, 1983; Ratner and Stratton, 1985) have reported excellent results from Thermonuclease, we found the test to lack both sensitivity and specificity on blood culture pellets. In addition, this test requires a boiling step and quality control is complicated. In reviewing rapid techniques for the identification of bacteria in blood cultures, Todd (1983) suggested methods should be "reproducible, objectively interpretable, and clinically relevant." The methods reported herein clearly meet the first two criteria for direct identification of S. aureus and St. pneumoniae. Shortening the time for diagnosis will allow more simplified and potentially less toxic antimicrobial therapy in bacteremic patients. We have demonstrated that S. aureus and St. pneumoniae may be identified directly on blood culture broth aliquots without the need for centrifugation and other separation techniques. Also, we have reduced the risk of needles and other contaminated devices by using blood culture vents to obtain a sample. These methods require no sophisticated instrumentation and they are simple, inexpensive, and accurate. Based on the results of this study, we have incorporated the tube coagulase and Pneumoslide tests into the routine blood culture workflow (Figure 1). Positive tests on BCBA are reported as "presumptive S. aureus" or "presumptive St. pneumoniae" and negative tests reported as "Gram-positive cocci." Subcultures are performed on blood agar and chocolate agar plates routinely for definitive identification and to rule out polymicrobic bacteremia. We gratefully acknowledge Patricia Lineback, Marsha Milish, and Sharmi Patel for their technical assistance, Joseph Brickler and James W. Smith for their review of the manuscript, and Beverly Jarrett for her secretarial assistance.
Identification of Staphylococcus aureus and Streptococcus pneumoniae
299
Positive Blood Culture
I
Gram Stain
Gram-positive cocci
Equivocal
Gram-positive cocci
in clusters
in pairs or chains
t~-"IA
I
2-hr tube coagulase I
Pneumoslide
I
I
+I
+
Jport Pres~.ptive S. aureus
f
BAP
I
I
P~port
Report
Report
Gram-positive
Prest.aptive
Gram-positive
cocci
St. pneumoniae
tt~
~
I
cocci
BAP
FIGURE 1 Flow diagram of the workflow used at Wishard Memorial Hospital when Gram-positive cocci are present in Gram stains of positive blood cultures (based on the results of this study). BAP, blood agar plate; and BCBA, blood culture broth aliquot.
REFERENCES Agger WA, Maki DG (1978) Efficacy of direct Gram stain in differentiating staphylococci from streptococci in blood cultures positive for Gram-positive cocci. J Clin Microbiol 7:111-113. Bryan CS (1989) Clinical implications of positive blood cultures. Clin Microbiol Rev 2:329-353. Dunagan WC, Woodward RS, Medoff G, Gray III JL, Casabar E, Smith MD, Lawrenz CA, Spitznagel E (1989) Antimicrobial misuse in patients with positive blood cultures. Am J Med 87:253-259. Facklam RR, Carey RB (1985) Streptococci and aerococci. In Manual of Clinical Microbiology, 4th ed. Eds, EH Lennette, A Balows, WJ Hausler Jr, and HJ Shadomy. Washington, DC: American Society for Microbiology, pp 154-175. Faruki H, Murray P (1986) Medium dependence for rapid detection of thermonuclease activity in blood culture broths. J Clin Microbiol 24:482-483. Fidalgo S, Vasquez F, Mendoza MC, Perez F, Mendez FJ (1990) Bacteremia due to Staphylococcus epidermidis: microbiologic epidemiologic, clinical, and prognostic features. Rev Infect Dis 12:520-528. Flejzor B, Bokkenheuser VD (1984) Use of Directogen and
Acridine Orange for rapid identification of human blood culture isolates. ] Clin Microbiol 20:587-588. Gordon LP, Datum MAS, Anderson JD (1987) Rapid presumptive identification of streptococd directly from blood cultures by serologic tests and the L-pyrrolidonyl-Bnaphthyl-amide reaction. J Clin Microbiol 25:238-241. Hamoudi AC, Marcon MJ, Cannon HJ (1984) Evaluation of rapid identification of Gram-positive cocci in positive blood cultures by use of the AutoMicrobic system grampositive identification card. J Clin Microbiol 20:171-174. Hamoudi AC, Hribar MM (1988) Evaluation of a direct identification method for Staphylococcusaureus from blood culture broth. J Clin Microbiol 26:1405. Hendrickson DA (1985) Reagents and stains. In Manual of Clinical Microbiology, 4th ed. Eds, EH Lennette, A Balows, WJ Hausler Jr, and HJ Shadomy. Washington, DC: American Society for Microbiology, pp 1093-1094. Kloos WE, Jorgenson JH (1985) Staphylococci. In Manual of Clinical Microbiology, 4th ed. Eds, EH Lennette, A Balows, WJ Hausler Jr, and HJ Shadomy. Washington, DC: American Society for Microbiology, pp 143-153. Knight RG, Shlaes DM (1983) Rapid identification of Staph-
300
ylococcus aureus and Streptococcus pneumoniae from blood cultures. J Clin Microbiol 17:97-100. Krumholz HM, Sande MA, Lo B (1989) Community-acquired bacteremia in patients with acquired immunodeficiency syndrome: clinical presentation, bacteriology and outcome. Am J Med 86:776-779. Kunin CM, Johansen KS, Worning AM, Daschner FD (1990) Reports of a symposium on use and abuse of antibiotics worldwide. Rev Infect Dis 12:12-19. Madison BM, Baselski VS (1983) Rapid identification of Staphylococcus aureus in blood cultures by thermonuclease testing. J Clin Microbiol 18:722. Murray PR (1979) Modification of the bile solubility test for rapid identification fo Streptococcus pneumoniae. J Clin Microbiol 9:290-291. Rappaport T, Sawyer KP, Nachamkin I (1988) Evaluation of several commercial biochemical and immuunologic methods for rapid identification of Gram-positive cocci directly from blood cultures. J Clin Microbiol 26:13351338. Ratner HB, Stratton CW (1985) Thermonuclease test for
T.E. Davis et al.
same-day identification of Staphylococcus aureus in blood cultures. J Clin Microbiol 21:995-996. Sahm DF, Kissinger J, Gilmore MS, et al. (1989) In vitro susceptibility studies of vanconycin-resistant Enterococcus faecalis. Antimicrob Agents Chemother 33:1588-1591. Severance PJ, Kauffman CA, Sheagren JN (1980) Rapid identification of Staphylococcus aureus by using lysostaphin sensitivity. ] Clin Microbiol 11:724. Shlaes DM, Toossi L, Patel A (1984) Comparison of latex agglutination and immunofluorescence for direct Lancefield grouping of streptococci from blood cultures. J Clin Microbiol 20:195-198. Strand CL, Shulman JA (1988) Bloodstream Infections: Laboratory Detection and Clinical Considerations. Chicago: American Society of Clinical Pathology, pp 36-38. Todd JK (1983) Nonculture techniques using blood specimens for the diagnosis of infectious disease. Am J Med 42:37-43. Uttley AHC, Collins CH, Naidoo J, et al. (1988) Vancomycin°resistant enterococci. Lancet 1:57-58.
295
Rapid, Direct Identification of Staphylococcus aureus and Streptococcus pneumoniae from Blood Cultures Using Commercial Immunologic Kits and Modified Conventional Tests Thomas E. Davis, DeAnna D. Fuller, and Ellen C. Aeschleman
To develop safe and rapid methods for identification of Staphylococcus aureus and Streptococcus pneumoniae directly from positive blood culture bottles (BCB) (BACTEC,Johnston Laboratories), several commercial biochemical and immunological tests as well as modified conventional tests were evaluated. Preliminary studies demonstrated that both S. aureus and St. pneumoniae could be identified directly using only a small aliquot (100 tzl) of the blood culture broth obtained via vent without need for centrifugation or other separation steps. A simple tube coagulase exhibited 98% sensitivity and 100%
specificity for 32 S. aureus isolates and 157 blood cultures positive for coagulase-negative staphylococci when read at 2 hr. All systems employed for direct identification of St. pneumoniae exhibited excellent sensitivity and specificity using " aliquots from blood culture broths, but Pneumoslide (BBL Microbiology Systems, Cockeysville, MD) was easiest to perform and interpret. The results of this study show that S. aureus and St. pneumoniae can be identified directly from blood culture broth aliquots using rapid methods that eliminate the need for centrifugation or use of needles and syringes.
INTRODUCTION
tients (Fidalgo et al., 1990). Early identification of staphylococci as coagulase negative may prevent unnecessary antibiotic therapy (Dunagan et al., 1989; Kunin et al., 1990). Bacteremias caused by S. aureus and St. pneumoniae require prompt and appropriate antibiotic therapy. They account for 50% of community-acquired bacteremias in AIDS patients, and rapid diagnosis can optimize therapy (Krumholz et al., 1989). Other Gram-positive cocci, especially enterococci, are often resistant to multiple antibiotics, and rapid identification guides choice of therapy (Sahm et al., 1989; Uttley et al., 1988). The direct identification of blood culture isolates has recently been reviewed by Strand and Shulman (1988) and, in most situations, agreement between rapid and conventional methods has been good. Rapid commercial tests have been applied directly to bacteria from blood cultures (Flejzor and Bokkenheuser, 1984; Hamoudi et al., 1988; Rappaport et al., 1988; Shlaes et al., 1984). Methods for rapid identification
The most common blood culture isolates at this and other institutions, are Gram-positive cocci (Bryan 1989; Rappaport et al., 1988). Among the Gram-positive cocci, coagulase-negative staphylococci, Staphylococcus aureus, and Streptococcus pneumoniae have been most frequent. Coagulase-negative staphylococci are frequently found in blood cultures as contaminants from the skin, but may be pathogenic in some paFrom the Wishard MemorialHospital--IndianaUniversity Medical Center (T.E.D., D.D.F., E.C.A.), Indianapolis, Indiana; and Watauga Hospital Inc. (E.C.A.), Boone, North Carolina, USA. Address reprint requests to Dr. T.E. Davis, Wishard Memorial Hospital, Department of Pathology, 1001 West 10th Street, Indianapolis, IN 46202, USA. Received 28 March 1991; revised and accepted 19 August 1991. © 1992 ElsevierScience Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00
296
of Gram-positive cocci have included Gram stain, coagglutination, and latex agglutination (Agger and Maki, 1978; Rappaport et al., 1988; Severance et al., 1980). We decided to compare both commercial immunologic identification systems and conventional modified laboratory tests for direct and rapid identification of blood culture isolates of S. aureus and St. pneumoniae. We arbitrarily chose 2 hr as the maximum time for completion of these tests.
MATERIALS AND METHODS Processing Positive Blood Cultures Blood cultures collected from patients at Wishard Memorial Hospital, Indianapolis, Indiana, were analyzed from January 1989 to June 1990. Blood (3-5 ml) was collected into BACTEC6A (NR) and 7A (NR) and/or 16 (NR) and 17 (NR) blood culture bottles (BCB) (Johnston Laboratories, Towson, MD). A blood culture bottle (BCB) with a growth index of 30 or greater by the BACTEC method was Gram-stained and if Gram-positive cocci were observed, then the Gram smear was categorized into one of three groups: Staphylococcus-like, Streptococcus-like, or equivocal.
Identification of Isolates Gram-positive cocci isolated from blood cultures were identified by standard laboratory methods (Facklam and Carey, 1985; Kloos and Jorgenson, 1985; Hendrickson, 1985). For presumptive identification of Gram-positive cocci directly from blood culture bottles, several methods were employed. Conventional tests included Gram's stain, slide bile solubility, NaC1Esculin test, slide coagulase, and the tube coagulase. Commercial kits evaluated were Staphaurex, Streptex, and Wellcogen Strep Test (Wellcome Diagnostics, Research Triangle Park, NC), Staphyloslide and Pneumoslide (BBL Microbiology Systems, Cockeysville, MD), and Thermonuclease Agar (Remel Laboratories, Lenexa, KS). A 24-hr tube coagulase test was performed on all microorganisms that tested negative for S. aureus at 2 and 4 hr (Kloos et al., 1985). Tests used for identification of St. pneumoniae included the slide modification of the bile solubility test, Pneumoslide, and Wellcogen Strep Test. Microorganisms described morphologically as streptococci that tested negative for St. pneumoniae were tested with Streptex, Fluorescent Antibody method for Group A Streptococcus, a 4-hr NaCI-Esculin broth, and Identicult A-E (Facklam and Carey, 1985). Tests for both staphylococci and streptococci were performed when the smear showed Gram-positive cocci without a predominance of clumps or chains.
T.E. Davis et al.
Polymicrobial bacteremia was rare and not included in this study. For identification of staphylococci and streptococci, three different sample types were used: (a) one drop (-100 ~I) of a well-mixed blood culture broth aliquot (BCBA) taken directly from the positive blood culture bottle using a blood culture bottle vent/subunit (Difco Laboratories, Detroit, MI); (b) 4-5 bacterial colonies representing colonial growth (CG) from agar plates inoculated from positive blood culture bottles and incubated 24 hr at 35°C; and (c) blood culture pellet (BCP) of bacteria collected from the positive blood culture bottles. The pellets were obtained by removing 10 ml of broth from the blood culture bottle and centrifuging at 150 g for 10 min to sediment out the red blood cells. The supernatant was then centrifuged at 1000 g to concentrate the bacteria. The resulting supernatant was removed, and the pellet was resuspended in 1 ml of sterile saline. For all three sample types, specific manufacturer's instructions were followed for test performance. For immunologic identification of staphylococci and streptococci, one drop of BCP, CG, or BCBA was transferred to a well on a test card (supplied with each reagent), and instructions of the manufacturers were followed. For biochemical identification of staphylococci and streptococci, one drop of BCP, CG, or BCBA was transferred to each biochemical tube or plate, and specific procedural instructions for our laboratory were followed.
RESULTS Direct Testing of Staphylococci from Blood Culture Pellets A total of 72 pellets containing staphylococci were examined by a variety of commercial immunological, biochemical, and rapid modifications of conventional identification methods (Table 1). The tube coagulase identified 100% of blood culture bottles containing S. aureus within 2 hr. The sensitivity was 68% with Thermonuclease Agar. The specificity for all methods ranged from 93% (Thermonuclease Agar) to 100% (tube coagulase).
Testing of Staphylococci from Corresponding Colonial Growth A total of 70 isolates obtained from blood culture pellets were subsequently tested with the same combination of tests performed from colonies on solid media on the following day (Table 1). The sensitivities were 100% for all tests, and the specificity ranged from 93% (Staphaurex) to 100% (Slide coagulase, Thermonuclease Agar, Staphyloslide, and tube coagulase).
297
Identification of Staphylococcus aureus a n d Streptococcus pneumoniae
TABLE 1
Identification of Staphylococcus aureus a n d Coagulase-Negative Staphylococci No. of Correct Identifications a
Specimen
Test System
Coagulase-Negative S. aureus Staphylococci
Sensitivity (%)
Specificity (%)
Negative Predictive Value (%)
Positive Predictive Value (%)
BCPb
Staphaurex Slide coagulase Thermonuclease Staphyloslide Tube coagulasec
9/24 12/24 15/22 8/24 24/24
47/48 46/47 41/44 45/46 47/47
38 50 68 33 100
98 98 93 98 100
76 79 85 74 100
90 92 83 89 100
CGa
Staphaurex Slide coagulase Thermonuclease Staphyloslide Tube coagulase
24/24 4/4 24/24 5/5
43/46 11/11 8/8 46/46 11/11
100 100 100 100 100
93 100 100 100 100
100 100 100 100 100
89 100 100 100 100
Staphaurex Slide coagulase Thermonuclease Staphyloslide Tube coagulasec
4/5 5/5 4/4 4/5 31/32
12/13 11/12 8/9 11/12 157/157
80 100 100 80 97
92 92 89 92 100
92 100 100 92 98
80 83 80 80 100
BCBAb
5/5
aThe first number represents correct identifications by the listed method and the second number represents the total number of isolates tested. bBlood cultures (18) containing staphylococci were tested using BCBA(blood culture broth aliquot) only. BCP, blood culture pellet. CAllbut one of the S. aureus isolates were coagulase positive within 2 hr. dColonial growth.
Direct Testing for Staphylococci Using 100-1~1 Blood Culture Broth Aliquots Isolates (189) were tested with the previously listed m e t h o d s (Table 1). The sensitivity r a n g e d from 80%
TABLE 2
(Staphaurex a n d Staphyloslide), to 97% (tube coagulase), to 100% (Slide coagulase a n d T h e r m o n uclease Agar). The specificity r a n g e d from 89% (Thermonuclease Agar) to 92% (Staphaurex, slide coagulase, Staphyloslide) to 100% (tube coagulase).
Identification of Streptococcus pneumoniae a n d from Blood Culture Pellets No. of Correct Identifications a
Specimen
Test System
Other St. pneumoniae Streptococci
Sensitivity (%)
Specificity (%)
Negative Predictive Value (%)
Positive Predictive Value (%)
BCPb
Pneumoslide Bile solubility Wellcogen strep test
23/23 24/24 20/24
19/19 17/17 13/13
100 100 83
100 100 100
100 100 76
100 100 100
CGC
Pneumoslide Bile solubility Wellcogen strep test
22/22 8/8 8/8
13/13 3/3 3/3
100 100 100
100 100 100
100 100 100
100 100 100
BCBAb
Pneumoslide Bile solubility Wellcogen strep test
32/32 23/23 25/26
40/40 5/5 5/5
100 100 96
100 100 100
100 100 83
100 100 100
aThe first number represents correct identifications by the listed method and the second number represents the total number of isolates tested. bBlood cultures (30) containing streptococci were tested using BCBA(blood culture broth aliquot) only. BCP, blood culture pellet. CColonygrowth.
298
Direct Testing of Streptococcus pneumoniae from Blood Culture Pellets Tests for St. pneumoniae were conducted on 42 BCP by using the slide modification of the bile solubility test, Pneumoslide, and Wellcogen St. pneumoniae latex agglutination (Table 2). The slide bile solubility and Pneumoslide yielded sensitivities of 100%, and the Wellcogen latex agglutination had a sensitivity of 83%. Each method yielded a specificity of 100%.
Testing of Streptococcus pneumoniae Using Corresponding Colonial Growth The above three methods were performed the following day on subsequent colonial growth (Table 2). The sensitivity and specificity of each was 100%.
Direct Testing for Streptococcus pneumoniae Using 100-~1 Blood Culture Broth Aliquots This additional procedure was performed using the slide bile solubility and Pneumoslide tests with both yielding sensitivities and specificities of 100% (Table 2). DISCUSSION Our study shows that rapid conventional methods (tube coagulase and slide bile solubility) can be used to identify S. aureus and St. pneumoniae directly from positive blood cultures. The sensitivity and specificity of these inexpensive tests match or surpass the more expensive commercial immunologic or biochemical kits evaluated simultaneously. These simple methods are accurate w h e n performed directly on blood culture broth aliquots and appear to be at least as reliable as those performed on colonial growth or blood culture pellets. As reported by others (Knight and Shlaes, 1983; Rappaport et al., 1988), the slide bile solubility test for St. pneumoniae is accurate but time consuming, as it requires an additional Gram stain and microscopic interpretation. We did not find false-positive tests with this method as experienced by others (Murray 1979). The slide bile solubility test is reliable for testing CG, BCP, and BCBA. We evaluated the Pneumoslide and Wellcogen Strep Test commercial kits and, as has been the experience of others (Gordon et al., 1987; Rappaport et al., 1988), these tests showed excellent sensitivity and specificity w h e n performed on blood culture broth aliquots. Pneumoslide was equivalent to the slide bile solubility test in accuracy and is easier
T.E. Davis et al.
to perform and interpret by our technologists. In our hands, the Pneumoslide test yielded 100% sensitivity and specificity on all specimens. Hamoudi and Hribar (1988) reported numerous false-positive and false-negative results with Staphaurex when attempting to identify S. aureus in blood culture pellets. In agreement with Rappaport et al. (1988), we found Staphaurex and Staphyloslide to be reliable for testing colonies from solid media, but both lacked sensitivity w h e n testing blood culture pellets. The tube coagulase test exhibited 100% specificity and failed to detect only one S. aureus. Further, the tube coagulase is simple to perform and fits well into the workflow of our blood culture bench. The slide coagulase method also works well, but requires more hands-on time. Although others (Faruki and Murray, 1986; Madison and Baselski, 1983; Ratner and Stratton, 1985) have reported excellent results from Thermonuclease, we found the test to lack both sensitivity and specificity on blood culture pellets. In addition, this test requires a boiling step and quality control is complicated. In reviewing rapid techniques for the identification of bacteria in blood cultures, Todd (1983) suggested methods should be "reproducible, objectively interpretable, and clinically relevant." The methods reported herein clearly meet the first two criteria for direct identification of S. aureus and St. pneumoniae. Shortening the time for diagnosis will allow more simplified and potentially less toxic antimicrobial therapy in bacteremic patients. We have demonstrated that S. aureus and St. pneumoniae may be identified directly on blood culture broth aliquots without the need for centrifugation and other separation techniques. Also, we have reduced the risk of needles and other contaminated devices by using blood culture vents to obtain a sample. These methods require no sophisticated instrumentation and they are simple, inexpensive, and accurate. Based on the results of this study, we have incorporated the tube coagulase and Pneumoslide tests into the routine blood culture workflow (Figure 1). Positive tests on BCBA are reported as "presumptive S. aureus" or "presumptive St. pneumoniae" and negative tests reported as "Gram-positive cocci." Subcultures are performed on blood agar and chocolate agar plates routinely for definitive identification and to rule out polymicrobic bacteremia. We gratefully acknowledge Patricia Lineback, Marsha Milish, and Sharmi Patel for their technical assistance, Joseph Brickler and James W. Smith for their review of the manuscript, and Beverly Jarrett for her secretarial assistance.
Identification of Staphylococcus aureus and Streptococcus pneumoniae
299
Positive Blood Culture
I
Gram Stain
Gram-positive cocci
Equivocal
Gram-positive cocci
in clusters
in pairs or chains
t~-"IA
I
2-hr tube coagulase I
Pneumoslide
I
I
+I
+
Jport Pres~.ptive S. aureus
f
BAP
I
I
P~port
Report
Report
Gram-positive
Prest.aptive
Gram-positive
cocci
St. pneumoniae
tt~
~
I
cocci
BAP
FIGURE 1 Flow diagram of the workflow used at Wishard Memorial Hospital when Gram-positive cocci are present in Gram stains of positive blood cultures (based on the results of this study). BAP, blood agar plate; and BCBA, blood culture broth aliquot.
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