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Parker agar medium (BPA) is commonly used for selective isolation of S. aureus, and the coagulase test is normally used for
identification of suspect colonies on this selective medium (1
3). Although the coagulase test is simple and reliable, falsepositive results can be caused by other Staphylococcus species
(4), and different animal plasma used for the test may result in
different results (5). Some citrate-utilizing bacteria may also
cause false-positives results, because citrate is one of the anticoagulants used during the collection of animal blood (6).
Moreover, for performing the coagulase assay, suspect isolates
on BPA need to be transferred to brain-heart infusion (BHI)
broth for overnight incubation; the culture broth is then tested
for coagulase activity, a step that may require a period as long
as 6 h. Because BPA is generally used to isolate S. aureus from
foods (13), a rapid identification method of the bacterium on the
medium may save 2 days comparing to the coagulase procedure.
Since the development of a slide agglutination test with
plasma-coated latex for rapid identification of S. aureus (7), several researchers (811) have evaluated the efficacy of some commercial latex agglutination kits for identification of clinical isolates
of S. aureus. Generally, favorable results have been obtained. The
latex agglutination test is very simple to perform, and results can
be obtained within minutes. However, none of these commercial
latex kits used for identifying S. aureus has been collaboratively
compared with the conventional coagulase test.
A latex agglutination kit (Aureus Test, Trisum Corp., Taipei,
Taiwan) for rapid identification of S. aureus recently became
commercially available. Anti-protein A immunoglobulin G
(IgG) and fibrinogen are used to coat polystyrene latex beads
to simultaneously bind protein A and coagulase, both of which
are specific cell surface components of S. aureus.
A precollaborative study (12) using 267 strains (of which
157 were S. aureus) was performed to compare the Aureus Test
latex agglutination method with AOAC Official Method 987.09
(conventional coagulase test). For the latex test, high sensitivity
(100%) and specificity (>94.4%) were obtained. The results
were comparable with or even better than those obtained with
the conventional coagulase test.
The objective of this study was to collaboratively compare
the Aureus Test kit with the AOAC Official Method 987.09
Statistical Analysis
Data from all collaborators were collected and statistically
analyzed. For S. aureus, qualitative results were analyzed by
using a pair-wise comparison of the rate of false-negative reactions by each method (latex agglutination versus coagulase) as
described by McNemar (13), according to the relationship:
2 =
(a b 1)2
a+b
tests positive by coagulase method and negative by latex agglutination method. A 2 value of >6.63 indicates significance at p
< 0.01.
For non-S. aureus bacteria, results were statistically analyzed by using a pair-wise comparison of the rate of false-positive reactions by each method (latex agglutination versus coagulase) as described by McNemar (13), according to the same
equation as above.
Sensitivity and specificity rates for both methods were calculated according to the method of McClure (14). Sensitivity is
defined as the number of test positives for S. aureus bacteria
(i.e., true positives) divided by total number of S. aureus tested.
Specificity is defined as the number of test negatives of non-S.
aureus (true negatives) divided by total number of non-S.
aureus bacteria tested. For latex agglutination, occasionally,
autoagglutination (nonspecific agglutination with both control
and test latex reagents) was observed. Strains showing autoagglutination were recorded as uninterpretable; these tests were
invalid and the data were excluded from statistical analysis.
995.12 Staphylococcus aureus Isolated from Foods,
Latex Agglutination Test Method
First Action 1995
(Method is agglutination test for rapid identification of
Staphylococcus aureus isolates from foods.)
Caution: See Appendix for Laboratory Safety for Safe
Handling of Microorganisms. Perform latex test in biosafety
cabinet (e.g., with laminar flow). Latex reagents contain 0.1%
sodium azide. Avoid contact with skin and eyes. In case of contact, immediately flush contact surfaces with plenty of water.
Disposal of this reagent into sinks with copper or lead plumbing should be followed immediately with large quantities of
water to prevent potential explosive hazards. Reagents are
harmful if swallowed. Autoclave all materials after use.
Method Performance:
See Tables 995.12AC for method performance data.
A. Principle
Bacteria are isolated on Baird-Parker agar medium and then
subcultured onto tryptic (trypticase) soy agar. Individual suspect colonies are transferred to reaction card and mixed with
polystyrene latex particles. Latex particles are coated with antiprotein A immunoglobulin G and fibrinogen that bind protein
A and coagulase (both are specific cell surface components of
S. aureus). Agglutination reaction is observed within ca 1 min
when S. aureus is present.
B. Apparatus
(a) Disposable reaction card.Contains 5 control circles
and 5 test circles. Card can be used for 5 tests.
(b) Disposable wooden applicator stick.100 pieces.
Items (a) and (b) are supplied in the Aureus Test kit from
Trisum Corp., Taipei, Taiwan.
F. Reading
Test is positive if agglutination is observed in test circle and
suspension in control circle remains homogeneous.
Test is negative if no agglutination is observed in test circle.
Test is uninterpretable if agglutination is observed in both
control and test circles. Strain showing uninterpretable latex
reaction must be confirmed by conventional tests as described
in 987.09 or in Bacteriological Analytical Manual, current edition, AOAC INTERNATIONAL, Arlington, VA.
Ref.: J. AOAC Int. 79, 661(1996)
Results and Discussion
Fifteen laboratories participated in the collaborative
study. Each laboratory tested 240 bacterial strains (Table 1).
In general, similar results were obtained whether test cultures were grown on BPA or TSA. Most strains of S. aureus
produced positive coagulase and latex agglutination reactions as expected. However, strains of S. aureus showing
false-negative reactions on TSA (Table 2) and BPA (Table 4)
were found. Tables 3 and 5 show strains of non-S. aureus
bacteria producing false-positive reactions on TSA and BPA,
respectively. Some uninterpretable reactions are also shown
in these tables.
False-negatives by the latex test (Tables 2 and 4) were
caused mainly by culture No. 183 (S. aureus ATCC 27703); 14
of 15 collaborators reported negative results for this strain. Cultures No. 62 (ATCC 14154), 161 (CCRC 13824), and 221
(CCRC 14834) mainly were responsible for false-negative coagulase tests. Thirteen collaborators obtained negative coagulase results for culture No. 221.
False positives with the latex test were found with cultures
No. 56, 59, 70, 188, and 197 (Tables 3 and 5). Occasionally,
autoagglutination was found when performing the latex agglutination assay. This phenomenon was reported also in other
studies (10, 12). It may be caused by the surface characteristics
of some bacteria. If autoagglutination occurs, the test becomes
uninterpretable and is invalid. Under this condition, the conventional identification procedure (13) should be followed.
However, the rate of autoagglutination was low (about 0.3%),
and only one-third (0.1%) was caused by S. aureus.
All 15 collaborators reported false-positive coagulase tests
for cultures No. 47 (S. intermedius ATCC 29663), 70 (S. intermedius ATCC 49052), 149 (S. delphini ATCC 49171), 150 (S.
delphini ATCC 49172), and 172 (S. intermedius ATCC 49051).
The ability of S. intermedius to clot plasma has been well documented in Bergeys Manual of Systematic Bacteriology (15).
Recently, S. delphini was reported to be coagulase positive
(16). A recent survey (4) found that S. intermedius and S. delphini are strong coagulase producers. Other false positives for
the coagulase test were found randomly in some strains of other
bacteria.
Method performance for bacteria grown on TSA is shown
in Table 995.12A. For the latex agglutination assay, among the
(11) Stevens, M., & Geary, C. (1989) Eur. J. Clin. Microbiol. Infect. Dis. 8, 153156
(12) Chang, T.C., & Huang, S.H. (1993) J. Food Prot. 56, 759762
(13) Siegel, S. (1956) Nonparametric Statistics for the Behavioral
Sciences, McGraw-Hill Book Co., New York, NY
(14) McClure, F.D. (1990) J. Assoc. Off. Anal. Chem. 73, 953960
(15) Kloos, W.E., & Schleifer, K.H. (1986) In Bergeys Manual of
Systematic Bacteriology, Vol. 2, P.H.A. Sneath, N.S. Mair,
M.E. Sharpe, & J.G. Holt (Eds), Williams & Wilkins Co.,
Baltimore, MD, pp. 10131035
(16) Varaldo, P.E., Kilpper-Balz, R., Biavasco, F., Satta, G., &
Schleifer, K.H. (1988) Int. J. Sys. Bacteriol. 38, 436439
Table 995.12A. Method performance for identification of Staphylococcus aureus isolated on tryptic soy agar
Test results
Microorganism
Total
Positives
Negatives
Sensitivity, %
Specificity, %
3
7
99.2
97.1
98.4
92.5
S. aureus
Other
2400
1200
2379
35
18
1158
AOAC Official Method 987.09F
S. aureus
Other
a
2400
1200
2362
90
38
1110
Table 995.12B. Method performance for identification of Staphylococcus aureus isolated on Baird-Parker agar
Test results
Microorganism
Total
Positives
Negatives
Sensitivity, %
Specificity, %
3
6
99.2
96.6
98.3
91.3
S. aureus
Other
2400
1026
2379
35
18
985
AOAC Official Method 987.09F
S. aureus
Other
a
2400
1026
2360
89
40
937
a Valuea
2 Value
b Valueb
S. aureus
Other
33
22
14
77
6.89
29.45
S. aureus
Other
35
22
14
76
8.16
28.66
0.01
S. aureus
S. auricularis
S. capitis subsp. capitis
S. capitis subsp. ureolyticus
S. caseolyticus
S. cohnii subsp. cohnii
S. delphini
S. epidermidis
S. haemolyticus
S. hominis
S. intermedius
S. lentus
S. lugdunensis
S. sciuri subsp. sciuri
S. simulans
S. warneri
S. xylosus
E. coli
Listeria monocytogenes
Micrococcus luteus
Salmonella typhimurium
Rhodococcus equi
Total
a
b
Culture No.
1, 2, 3, 4, 5, 8, 9, 10, 13, 14, 15, 16, 17, 20, 21, 22, 25, 26, 27, 28,
31, 32, 33, 35, 36, 39, 40, 42, 44, 45, 46, 49, 50, 52, 53, 54, 55,
57, 58, 60, 61, 62, 63, 64, 67, 68, 69, 71, 72, 74, 75, 76, 78, 79,
80, 81, 84, 85, 87, 90, 91, 92, 93, 96, 97, 99, 100, 101, 102, 105,
106, 107, 108, 111, 112, 113, 114, 116, 117, 120, 121, 122, 123,
125, 126, 127, 130, 131, 133, 134, 135, 136, 139, 140, 141, 144,
145, 146, 147, 148, 151, 152, 153, 154, 156, 157, 158, 161, 162,
163, 166, 167, 169, 170, 171, 173, 174, 175, 178, 179, 181, 182,
183, 186, 187, 189, 190, 191, 194, 195, 196, 198, 199, 201, 202,
203, 205, 206, 207, 210, 211, 212, 214, 215, 216, 218, 219, 221,
222, 223, 226, 227, 228, 231, 232, 233, 234, 236, 237, 240
103, 104, 115, 164
12, 95
30, 109, 110
56, 59
43, 119, 124, 165, 188
149, 150
7, 128, 129, 132, 192, 213
18, 176, 177, 193, 217
83, 88, 89, 168, 200, 208
47, 70, 172
24, 209
29, 197
19, 180, 184, 224
6, 220, 230
48, 51, 118, 225, 229
23, 73, 82, 86, 185, 204
38, 41, 138
65, 66, 77, 94, 98
155, 159, 160, 235, 238
34, 37, 137
11, 142, 143, 239
Number of
isolates
Source
a
160
4
2
3
2
5
2
6
5
6
3
2
2
4
3
5
6
3
5
5
3
4
240
ATCC
CCRC, ATCC
ATCC
ATCC
CCRC, ATCC
ATCC
CCRC, ATCC
CCRC, ATCC
ATCC, CCRC
ATCC
ATCC, CCRC
ATCC, CCRC
ATCC, CCRC
CCRC
ATCC, CCRC
ATCC, CCRC
CCRC
ATCC, CCRC
ATCC
CCRC
CCRC
Table 2. Strains of S. aureus showing false-negative coagulase test or latex agglutination test or uninterpretable
reaction (cultures grown on TSA)
Coagulase test
Latex test
Collaborator
Culture
No.
9
46
53
61
62
145
151
161
183
216
221
222
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
a
b
10 11 12 13 14 15
FNa
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
10 11 12 13 14 15
FN
FN
FN
FN
FN
FN
FN
FN
Ub
U
U
Table 3. Strains of non-S. aureus bacteria showing false-positive coagulase test or latex agglutination test or
uninterpretable reaction (cultures grown on TSA)
Coagulase test
Latex test
Collaborator
Culture
No.
10 11 12 13 14 15
10 11 12 13 14 15
23
24
29
30
43
47
56
59
70
103
110
124
149
150
172
188
192
193
197
200
224
+
+
+
+
+a
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Ub
+
+
+
U
+
U
U
+
+
+
+
+
U
+
+
+
+
a
b
+, false positive.
U, uninterpretable latex reaction.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
U
+
Table 4. Strains of S. aureus showing false-negative coagulase test or latex agglutination test or uninterpretable
reaction (cultures grown on BPA)
Coagulase test
Latex test
Collaborator
Culture
No.
9
46
53
61
62
145
151
161
183
216
221
222
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
a
b
10 11 12 13 14 15
FNa
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
FN
10 11 12 13 14 15
FN
FN
FN
FN
FN
FN
FN
FN
Ub
U
U
Table 5. Strains of non-S. aureus bacteria showing false-positive coagulase test or latex agglutination test or
uninterpretable reaction or no growth (cultures grown on BPA)
Coagulase test
Latex test
Collaborator
Culture
No.
10 11 12 13 14 15
10 11 12 13 14 15
11
Na
23
b b
24
29
30
34
37
38
41
43
47
48
51
56
59
Uc
65
70
88
89
95
98
103
104
110
115
128
129
132
137
138
142
143
149
N
N
150
155
159
160
164
168
172
176
185
188
193
197
200
204
Table 5. (continued)
Coagulase test
Latex test
Collaborator
Culture
No.
10 11 12 13 14 15
10 11 12 13 14 15
208
224
235
238
239
a
b
c
N, no growth on BPA and was not tested by the coagulase method and the latex method.
+, false positive.
U, uninterpretable latex reaction.
+
N