BRIEF SCIENTIFIC REPORTS
Vol. 81 -No. 3
M. bovis is inhibited by TCH except for isoniazid-resistant
strains.7
The BACTEC system for recovery of mycobacteria,
although more costly than conventional procedure using
slanted media, offers valuable improvements in clinical
microbiology laboratory services. These benefits include
shorter time for recovery, separation of M. tuberculosis
from MOTT bacilli, and susceptibility testing of mycobacteria, in addition to a higher yield of positive cultures.
References
1. Cummings DM, Ristroph D, Camargo EE, Larson SM, Wagner
HN: Radiometric detection of the metabolic activity of Mycobacterium tuberculosis. J Nucl Med 1975; 16:1189-1191
2. Damato JJ, Rothlauf M, McClatchy JK: Differentiation of tubercle
3.
4.
5.
6.
7.
345
bacilli and non-tuberculous mycobacteria by a radiometric
method. Abstracts of the Annual Meeting of the American Society
for Microbiology 1980; C195:307
Kubica GP, Dye WE, Cohn ML, Middlebrook G: Sputum digestion
and decontamination with N-acetyl-L-cysteine-sodium hydroxide
for culture of mycobacteria. Am Rev Resp Dis 1963; 87:775779
Libonati JP, Siddiqi H, Carter M, Hwangbo C: Recovery and identification of mycobacteria from clinical specimens using radiometric methods. Abstr Annu Meet Am Soc Microbiol 1982;
CI 90:303
Middlebrook G, Reggiardo Z, Tigertt WD: Automatable radiometric
detection of growth of Mycobacterium tuberculosis in selective
media. Am Rev Resp Dis 1977; 115:1067-1069
Tahahaski H, Foster V: Detection and recovery of mycobacteria
by a radiometric procedure. J Clin Microbiol 1983; 17:380-381
Vestal AL: Procedures for the isolation and identification of mycobacteria. Center for Disease Control (DHEW) 1975; Publication
no. 76-8230:68
An Evaluation of Three Rapid Coagglutination Tests: SeroSTAT®, Accu-Staph™, and Staphyloslide™, for Differentiating
Staphylococcus aureus from Other Species of Staphylococci
BERT F. WOOLFREY, M.D., PH.D., RICHARD T. LALLY, M.S., M(ASCP), AND MARY NEILS EDERER, M.S.
Three commercial coagglutination tests—Sero-STAT®, AccuStaph™, and Staphyloslide™—were performed in parallel with
slide coagulase, tube coagulase, and thermostable nuclease tests
on 100 methicillin-susceptible Staphylococcus aureus (MSS)
strains, 100 methicillin-resistant S. aureus (MRS) strains, and
100 non-5, aureus staphylococcal strains (NSA). All three coagglutination tests showed sensitivities of 100% for MSS strains.
For MRS strains, sensitivities were, respectively, 99%, 100%,
and 99%. False-positive reactions were, respectively, 10%, 2%,
and 2%. A marked difference in slide coagulase test sensitivity
was found for MSS strains (79%) and MRS strains (14%).
These findings suggest that the coagglutination tests may be
less sensitive for detecting MRS strains than for detecting MSS
strains and that these properties may be related to clumping
factor reactivity. The high false-positive rate for Sero-STAT
and even the 2% false-positive rate for Accu-Staph and Staphyloslide make clinical usefulness at this time somewhat problematic and debatable. In view of these findings, the authors
prefer to retain the tube coagulase test and thermostable nuclease
test for differentiation of S. aureus from non-5, aureus strains
in their laboratory. (Key words: Staphylococcus aureus; Methicillin-resistant S. aureus; Coagglutination; Protein A; Clumping
factor) Am J Clin Pathol 1984; 81: 345-348
Received April 8, 1983; received revised manuscript and accepted for
publication September 12, 1983.
Funds for this study were provided by the St. Paul-Ramsey Hospital
Medical Education and Research Foundation, Grant No. 8333.
Address reprint requests to Dr. Woolfrey: Clinical Microbiology Section, Department of Anatomic and Clinical Pathology, St. Paul-Ramsey
Medical Center, 640 Jackson Street, St. Paul, Minnesota 55101.
Clinical Microbiology Section, Department of Anatomic and
Clinical Pathology, St. Paul-Ramsey Medical Center,
St. Paul, Minnesota
ALTHOUGH a variety of methods have been proposed
for differentiating Staphylococcus aureus from other species of staphylococci, only a few have found practical
application in the clinical laboratory. Detection of free
coagulase by the tube coagulase test is the procedure of
choice for most clinical laboratories, but disagreement
still exists relative to time and temperature of incubation,
definition of positive reactions, and choice of
plasma. 8 1 0 1 6 1 7 The detection of bound coagulase, or
clumping factor, by the slide coagulase test permits rapid
and relatively specific identification of the majority of S.
aureus strains, but this procedure must be used in conjunction with the tube coagulase test to assure detection
of all isolates. 13 The thermostable nuclease test, which
detects the presence of deoxyribonucleases, provides high
sensitivity and specificity but is not widely used in the
clinical setting because of time and manipulative constraints. 10,18 A welcome addition to the clinical laboratory
armamentarium would be the development of a rapid,
sensitive, specific, and cost-effective method for differentiating S. aureus from other species of staphylococci.
WOOLFREY, LALLY, AND EDERER
346
Towards this end, promising results have been reported
for the coagglutination procedures employing either sheep
red blood cells (SRBCs) or latex particles variously sensitized to react with specific cell components of S. aureus,
such as protein A and clumping factor.5,9 Although protein
A content varies considerably in strains of S. aureus, both
specificity and sensitivity of more than 98% have been
reported for approaches using SRBCs sensitized with antiSRBC IgG to detect protein A by reaction with the F c
portion of IgG.6 Similarly, although clumping factor
reactivity varies considerably among S. aureus strains,
sensitivities and specificities of more than 98% have been
reported for procedures using SRBCs sensitized with fibrinogen to detect clumping factor.2 Comparable results
also have been described for the use of latex particles
sensitized with both IgG and fibrinogen by plasma coating
so as to detect both protein A and clumping factor.5
At present, three commercial coagglutination tests for
S. aureus identification are available. Sero-STAT® (Scott
Laboratories, Inc., Fiskeville, RI) and Accu-Staph™
(Carr-Scarborough Microbiologicals, Inc., Stone Mountain, GA) use IgG and fibrinogen-sensitized plasma-coated
latex particles, and Staphyloslide™ (BBL Microbiology
Systems, Cockeysville, MD) uses fibrinogen-sensitized
SRBCs. Clinical laboratory evaluations of Sero-STAT applied to clinical isolates have found results that were comparable to those for the tube coagulase test. 4 " Recent
preliminary reports of Accu-Staph and Staphyloslide have
indicated results comparable to Sero-STAT. Methicillinresistant isolates, many strains of which in our experience
are deficient for clumping factor reactivity and therefore
potentially unreactive in some coagglutination systems,
were not examined specifically.'-3714 The present study
was designed to evaluate and compare the usefulness of
these three commercial coagglutination systems for differentiating strains of S. aureus, both methicillin-sensitive
and methicillin-resistant, from other staphylococci.
Materials and Methods
Experimental Design
Three commercial coagglutination systems for identification of 5. aureus—Sero-STAT, Accu-Staph, and
Staphyloslide—were tested in parallel using 100 strains
of methicillin-susceptible S. aureus (MSS), 100 strains of
methicillin-resistant S. aureus (MRS), and 100 non-5.
aureus staphylococcal strains (NSA). Isolates were retrieved from stock culture, randomized, coded, and submitted to parallel testing with single lots of the commercial
coagglutination tests and conventional microbiologic tests,
as described below. All isolates had been characterized
for slide and tube coagulase activity and for methicillin
susceptibility at the time of isolation in the clinical lab-
A.J.C.P. • March 1984
oratory. Isolates also were tested for slide coagulase, tube
coagulase, and thermostable nuclease in parallel with the
commercial coagglutination tests. Minimum inhibitory
concentration (MIC) values were available from a previous
study of the stock cultures. Isolates with commercial
coagglutination test results that differed from conventional
test results were retested by all three commercial tests
and by the slide coagulase, tube coagulase, and thermostable nuclease tests. Quality control was performed
daily on each new vial of test reagent for each system,
using known laboratory strains of 5. aureus and Staphylococcus epidermidis.
Microorganisms
Three hundred recent clinical isolates of staphylococci
were selected from the St. Paul-Ramsey Medical Center
Clinical Microbiology Laboratory stock culture collection
on the basis of their previous species identification by
standard microbiologic methods and for their MIC values,
as determined by agar dilution.12 Each isolate had been
characterized at the time of isolation in the clinical laboratory by Gram's stain, catalase reaction, slide coagulase
test, and tube coagulase test.8 Methicillin resistance was
defined as an MIC of >8 Mg/mL and methicillin susceptibility as <,% /ig/mL. At the time of retrieval from stock
culture, all isolates again were characterized by Gram's
stain, catalase test, slide coagulase test, tube coagulase
test, and a modification of the thermostable nuclease test.15
S. aureus isolates were defined as being gram-positive
cocci, catalase positive, slide and/or tube coagulase positive, and thermostable nuclease positive. NSA were defined as gram-positive cocci, catalase positive, slide and
tube coagulase negative, and thermostable nuclease negative. Each isolate was retrieved from - 7 0 ° C stock culture
by plating onto single plates of sheep blood agar, which
were incubated at 35°C in air for 18-24 hours. One colony
from each plate then was passaged to a single fresh sheep
blood agar plate and also to a tube containing 4 mL brain
heart infusion broth. These inoculated plates and tubes
were incubated at 35°C in air for 18-24 hours. Colonies
from the plates then were tested by the three commercial
coagglutination systems according to the manufacturers'
instructions. The brain heart infusion broth was used for
thermostable nuclease testing. Isolates with discrepant results, as described above, were evaluated by repeat testing
from a repeat stock culture retrieval.
Sero-STAT Test
Each Sero-STAT staphylococcus test kit contains vials
of plasma-coated latex particles suspended in phosphatebuffered saline, and vials of 0.85% saline, sufficient for
120 or 240 test determinations. To perform the test, one
drop of saline was placed on a precleaned glass slide and,
BRIEF SCIENTIFIC REPORTS
Vol. 81 -No. 3
347
Table 1. A Comparison of the Results of Three Commercial Rapid Slide Coagglutination Tests and Conventional
Microbiologic Tests for Differentiating Methicillin-susceptible and Methicillin-resistant Strains
of S. aureus from Other Species of Staphylococci
MSS
MRS
NSA
No.
Tested
Slide
Coagulase
Tube
Coagulase
Thermostable
Nuclease
100
100
100
79*
14
0
100
100
100
0
100
0
Sero-STAT
Accu-Staph
Staphyloslide
100
99
10
100
99
2
100
100
2
• Numbers relating to the various tests represent number of tests with positive reactions.
using a sterile inoculating wire, no less than five colonies
or a sweep of confluent growth of the test isolate were
emulsified into a smooth paste-like suspension. One drop
of resuspended plasma-coated latex particles then was
added and mixed into the suspension using a sterile applicator stick. The slide was rocked gently by tilting back
and forth for 45 seconds. The test was considered positive
when an agglutination pattern of large clumps was observed within 45 seconds by the unaided eye. A negative
test resulted in little or no appreciable clumping. If nonspecific clumping occurred with saline alone, the test was
considered uninterpretable.
Accu-Staph Test
The Accu-Staph latex slide agglutination kit contains
three vials of plasma-coated latex particles in buffered
saline, sufficient for 120 tests. One drop of 0.85% saline,
not provided by the manufacturer, was placed on a precleaned glass slide. Five or more colonies of the test isolate
were emulsified in the saline to result in a heavy, smooth
suspension. One drop of resuspended Accu-Staph plasmacoated latex particles then was added and mixed thoroughly using a sterile applicator stick. The slide was rocked
gently back and forth for 45 seconds to 1 minute. The
test was considered positive for S. aureus when clumps
of agglutination were visible within 1 minute by the unaided eye. A negative test resulted in little or no agglutination within 1 minute. If agglutination of the isolate
occurred in saline alone, the test was considered uninterpretable.
Staphyloslide Test
The BBL Staphyloslide test is a hemagglutination test
designed to detect S. aureus on the basis of clumping
factor reactivity. One 200-test kit contains one vial of
fibrinogen-sensitized SRBC as positive reagent and one
vial of nonsensitized SRBC as the negative control. If
clumping factor is present on the suspect isolate, reaction
of the fibrinogen-sensitized SRBC with clumping factor
causes fibrinogen polymerization and subsequent agglutination. For each test isolate, both the sensitized and
nonsensitized SRBC were mixed thoroughly but gently.
One drop each of positive and negative reagent then was
dispensed onto a precleaned slide. To each set of drops,
one to three colonies were added and thoroughly mixed
with a sterile inoculating wire. The slide was rocked gently
for 15 seconds and interpreted immediately for hemagglutination. A positive test resulted in a large amount of
clumping with the sensitized SRBCs within 15 seconds,
which was visible to the unaided eye, while the unsensitized SRBCs remained homogeneous. No visible
clumping in either reagent after 15 seconds was considered
to be a negative test. If clumping was observed in both
reagents, the test was considered uninterpretable.
Results
Table 1 summarizes the results of the commercial
coagglutination tests in comparison with the results of
conventional slide coagulase, tube coagulase, and thermostable nuclease tests. A marked difference in slide coagulase reactivity was found for MSS and MRS strains.
The coagglutination tests produced no false-negative results with MSS strains, but Sero-STAT and Accu-Staph
each produced one (1%) false-negative result for the MRS
strains. On repeat testing of the false-negative isolates,
the Sero-STAT result again was negative, whereas the
Accu-Staph result changed to positive. A false-positive
rate of 10% was observed for Sero-STAT in contrast to
a 2% false-positive rate for the other two coagglutination
tests. One strain was misidentified initially and on repeat
testing by all three systems. When the ten Sero-STAT
false-positive strains were retested by Sero-STAT, seven
remained as false-positive reactions, while three changed
to negative. Accu-Staph and Staphyloslide each produced
one additional false-positive strain. When these two isolates were retested by Sero-STAT, both were classified as
positive. On retesting by Accu-Staph, one of the two initially Accu-Staph false-positive strains was corrected to
negative and one of the ten strains that were falsely positive
by Sero-STAT was classified incorrectly. Staphyloslide
produced two false-positive tests initially but corrected
one on retesting. In addition, Staphyloslide produced one
uninterpretable result for the NSA group that also remained as uninterpretable in retesting. This isolate was
WOOLFREY, LALLY, AND EDERER
348
identified correctly by the other two systems in initial
testing but was falsely positive by both on repeat testing.
Discussion
In comparison with the tube coagulase test and thermostable nuclease test results for differentiating S. aureus
from other species of staphylococci, all three coagglutination tests showed sensitivities of 99% or more. The falsenegative results occurred only with MRS strains. This is
interesting in that a strikingly low incidence of positive
slide coagulase test reaction also was found for MRS
strains. Since the slide coagulase test is based on the detection of clumping factor, it is possible that the decreased
sensitivity for detecting MRS strains by the coagglutination tests also may be related to decreased cell wall
clumping factor. It is also conceivable that the observed
small differences in sensitivities may not be borne out if
very large numbers of isolates were tested. The false-positive rate of 10% for Sero-STAT is significantly larger than
that observed by other investigators4" and might be related to variations in reagent lots. This fairly large falsepositive rate would permit Sero-STAT to be used for S.
aureus screening but negate its use in the clinical laboratory for specific identification of S. aureus. Although
a much lower false-positive rate (2%) was found for AccuStaph and Staphyloslide, their use as appropriate identification systems for 5. aureus in the clinical laboratory
remains to us both problematic and debatable. Both AccuStaph and Sero-STAT changed an initially correct result
to an incorrect result on repeat testing, indicating the
potential for a significant deficiency in test reproducibility.
Although these currently available commercial coagglutination systems are rapid and relatively inexpensive, the
results of this investigation indicate to us that the tube
coagulase test and/or the thermostable nuclease test should
remain the tests of choice for S. aureus identification in
the clinical laboratory.
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