1. 검 사 원 리 - 라텍스 응집법 (Latex-Agglutination Method)
- 1 -
- Fibrinogen 과 immunoglobulin이 코팅된 라텍스 시약과 검체 중의 Staphlyococcus aureus의 표면항원
(clumping factor와 protein A)이 응집반응을 보인다.
- Clumping factor negative 균주의 대부분인 MRSA의 경우, 80% 이상에서 polysaccharide
capsule에 둘러싸여 있으며, 이러한 capsular serotype 5 항원에 직접 반응하는 monoclonal 항체가 라텍스
에 코팅되어 감도 및 검출률을 높였다.
- MSSA의 경우 100%, MRSA의 경우 99.3%의 감도를 보인다.
- 결과는 음성 대조액의 응집이 없는 상태에서 검체의 응집의 유무를 육안으로 관찰 판독하여 양성 또는
2. 검 체
1) 검체 종류 : 18-24시간 배양된 의심 균주 및 Gram 양성이면서 Catalase 양성인 균주
2) 검체량 : 1 집락 – 3 집락
(선택 또는 비선택 배지 모두 가능)
3. 시 약 – Pastorex Staph Plus
1) 라텍스 시약(dropper bottle) : Fibrinogen, IgG, capsular polysaccharide에 대한 단클론 항체가 코팅된 라
2) 음성 콘트롤(drepper bottle)
3) 1회용 카드 및 스틱
4. 기구 및 장비
1) 교반기 (Rotator) 2) 초시계
5. 검 사 방 법
1) 시약을 사용 전에 잘 흔들어 섞어준다.
2) 1회용 반응 카드에 라텍스 시약 및 음성 대조액을 각각 한 방울씩 떨어뜨린다.
3) 루프나 시약 내 스틱을 이용하여 배양 배지에서 자란 균주를 1-3 집락을 취해 10초간 섞어준다.
4) 음성대조액과도 마찬가지로 반응시킨다.
5) 반응 카드를 교반기 등을 통해 30초 정도 혼합한 후 응집 유무를 관찰한다.
6) 응집 유무에 따라 양성 또는 음성으로 판정한다.
6. 결과판독 및 보고방법
- 음성대조액의 응집이 없어야 한다.
- 2 -
- 음성대조액이 응집을 보이는 경우는 자가응집 균주로 예상되므로 다른 방법으로 확인한다.
- 검체의 응집이 없으면 “음성”
- 검체가 적색의 응집을 보이면 “양성”으로 보고한다.
7. 참 고 치
1) 정성검사 : 음성 대조액을 기준으로 응집 유무
8. 결과해석 및 임상적의의
1) Staphylococcus aureus 의 동정에 이용한다.
2) MSSA 뿐 아니라 MRSA에 대한 감지도 관찰 가능하다.
9. 검사시 유의사항
1) 새로 발견된 Staphylococcus lugdunensis 및 Staphylococcus schleiferi의 경우 fibrinogen 흡착 능력이
있어 clumping factor와 반응할 수 있다.
2) Stphylococcus saprophyticus 같은 몇몇 Staphylococcus 균종은 라텍스와 비특이적 반응의 원인으로
알려져 있으므로 매 검사시마다 대조액과 비교 검사를 추천한다.
3) Streptococci는 면역글로불린의 Fc fragment와 반응하는 단백질을 갖고 있어서 위양성을 보일 수 있으
며, E.coli나 Candida albicans 등의 균주가 비특이적 응집을 보일 수 있으나, 이는 검사 전에 Gram 염
색이나 Catalase 반응을 시켜봄으로써 방지할 수 있다.
4) 라텍스 성분과 반응하는 물질을 생성하지 않는 S.aureus나 부적절한 집락을 취했을 경우 위음성이 나
타날 수 있다.
10. 정 도 관 리
① 정도관리 물질 : Staphylococcus aureus(양성) 및 Staphylococcus epidermidis(음성)
② 정도관리 방법 : 대상 검체와 동일한 방법으로 시행한다.
2) 외부정도관리 : CAP (년 3회), 국내 정도관리 (년 2회)
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1996, p. 2267–2269 Vol. 34, No. 9
Copyright 1996, American Society for Microbiology
Comparison of Five Tests for Identification of
Staphylococcus aureus from Clinical Samples
AD LUIJENDIJK, ALEX VAN BELKUM, HENRI VERBRUGH, AND JAN KLUYTMANS*
Department of Bacteriology, University Hospital Rotterdam, 3015 GD Rotterdam, The Netherlands
Received 26 February 1996/Returned for modification 22 April 1996/Accepted 24 June 1996
Five different laboratory tests for the identification of Staphylococcus aureus were compared. Analyses of 271
presumptive S. aureus strains, supplemented with 59 well-defined methicillin-resistant S. aureus (MRSA)
isolates, were performed. Only the Staphaurex Plus (Murex Diagnostics, Dartford, United Kingdom) and the
Pastorex Staphplus (Sanofi, Marnes-La-Coquette, France) tests displayed 100% sensitivity. The observed
difference with the free-coagulase test (Bacto coagulase plasma; Difco, Detroit, Mich.), a bound-coagulase
(clumping factor) test, and the former Staphaurex test (Murex Diagnostics) was caused mainly by the inability
of these three tests to identify some MRSA strains correctly. Among Polish MRSA isolates included in the
analysis, a group of free-coagulase-negative S. aureus strains was detected. Genetic typing by random amplification
of polymorphic DNA revealed that the strains showing aberrant behavior when the different test results
were compared belonged to a limited number of S. aureus clones.
Staphylococcus aureus is one of the most frequently encountered
pathogens in clinical specimens. To make the distinction
between this species and other, less virulent, staphylococci it is
of importance to have a reliable, fast, simple, and cheap identification
test available. For many years the tube test, which
detects the production of free coagulase, was considered the
“gold standard” for this purpose (4). A more simple and widely
used test is the detection of bound coagulase (clumping factor),
for instance, by a slide agglutination reaction. This test,
however, is less reliable and can produce both false-positive
and false-negative results (4). In recent years, several commercial
agglutination assays which are based on the detection of
clumping factor and several other products specific for S. aureus
(e.g., protein A) have become available. In a previous
report it was shown that a significant number of clinical isolates
of methicillin-resistant S. aureus (MRSA) gave negative results
by one of these newer tests (Staphaurex; Murex Diagnostics
Limited, Dartford, United Kingdom) (2). Therefore, the manufacturer
made some modifications which were aimed at improving
the test’s sensitivity.
In this study we evaluated the former and current versions of
Staphaurex and compared them with three other diagnostic
tests. These assays are a free-coagulase test (Bacto coagulase
plasma; Difco Laboratories, Detroit, Mich.), a bound-coagulase
test, and the Pastorex Staphplus (Sanofi Diagnostics Pasteur,
SA, Marnes-La-Coquette, France) test. Besides clinical
specimens, an additional number of well-defined MRSA
strains were included.
MATERIALS AND METHODS
S. aureus strains. Consecutive clinical samples (n 271) processed in the
Department of Bacteriology of the University Hospital Rotterdam yielded colonies
suspected to be S. aureus isolates. This observation was based on morphological
criteria. In addition, 59 strains of MRSA were tested. These strains were
derived from different patients in several European countries. A relatively large
proportion of the MRSA strains came from Poland (n 31; 52.5%). To determine
methicillin susceptibility, a disk diffusion method on Mueller-Hinton agar
(CM337; Oxoid, Amsterdam, The Netherlands) using a 5-g methicillin disk
(Oxoid) was used (1). Breakpoints were determined according to the criteria of
* Corresponding author. Mailing address: Department of Clinical
Microbiology, Ignatius Hospital Breda, P.O. Box 90158, 4800 RK
Breda, The Netherlands. Phone: (31) 765258015. Fax: (31) 765225014.
the National Committee for Clinical Laboratory Standards (6). A total of 330
staphylococcal isolates were tested. In the end, the clinical samples yielded 241
S. aureus isolates derived from 158 patients. The number of isolates per patient
ranged from 1 to 10.
Tests for identification of S. aureus. (i) Free-coagulase (tube) test. Bacto
coagulase plasma (Difco) was used according to the manufacturer’s instructions.
Briefly, two to four colonies were suspended in 0.5 ml of coagulase plasma,
mixed, and incubated at 37C. The tubes were inspected for formation of a clot
every hour until 4 h had passed and then were inspected after 24 h. Positive and
negative control strains were included in every run.
(ii) Bound-coagulase (agar) test. For detection of clumping factor, an agar
plate containing human fibrinogen was used (10). A colony of staphylococci was
transferred from the original plate to the coagulase agar plate with a sterile loop.
The plate was incubated for 18 h at 37C and inspected immediately thereafter.
If a zone of opaqueness in the agar surrounding the colony was visible the test
was considered positive. In each run, positive and negative control strains were
(iii) Staphaurex and Staphaurex Plus (Murex Diagnostics Limited). Staphaurex
is a latex agglutination test which detects clumping factor and staphylococcal
protein A simultaneously. Staphaurex Plus is a newer version which has included
the detection of antigens raised against difficult-to-detect MRSA strains (2). The
procedures for both tests were identical except for the reaction time required,
being 20 s for Staphaurex and 30 s for Staphaurex Plus. One drop of test latex was
placed in one circle of the reaction card. The blunt head of a mixing stick was
covered with bacteria, and these were emulsified in the drop of test latex. Then,
the card was rocked slowly while agglutination was observed for. If agglutination
occurred within the required reaction time, the same procedure was repeated
with control latex to check for nonspecific agglutination. If this type of autoagglutination
occurred, the result was considered noninterpretable.
(iv) Pastorex Staphplus (Sanofi). The Pastorex Staphplus test is based on the
same principles as those of the Staphaurex tests, i.e., latex agglutination with
detection of clumping factor, staphylococcal protein A, and capsular polysaccharides
for the detection of certain MRSA strains (2). The test procedure was
identical to the procedure of the Staphaurex test.
Additional testing of strains with discordant results. (i) Retesting. Strains with
variable outcomes when the results of the different tests were compared were
retested by all procedures mentioned previously and were subsequently studied
further with the aid of the additional tests mentioned below. The Accuprobe
assay and the coagulase gene PCR can be considered the gold standard; all
strains ultimately tested were positively identified as S. aureus by both tests.
(ii) Free-coagulase tests. Three additional tube tests for detection of free
coagulase were used: first, the Bacto coagulase EDTA (Difco) test; second, the
Staphylocoagulase (Sanofi) test; and third, the rabbit plasma tube test developed
in our laboratory. The procedure for each test was identical to that of the Bacto
coagulase test described above.
(iii) Accuprobe (Gen-Probe Incorporated, San Diego, Calif.). The Accuprobe
S. aureus culture identification test detects specific rRNA sequences that are
unique to S. aureus. Therefore, it is considered to be independent from the
production of coagulase, clumping factor, and other phenotypic characteristics. It
uses a chemiluminescent DNA probe, and after hybridization a selection step
eliminates any unhybridized probe. The procedure was performed according to
the manufacturer’s instructions. Briefly, a loopful of bacteria was suspended in 50
jcm.highwire.org by on November 11, 2009
2268 LUIJENDIJK ET AL. J. CLIN. MICROBIOL.
TABLE 1. Survey of the results of the different S. aureus identification assays
l of lysis reagent and incubated for 5 min in a water bath at 37C. Thereafter,
50 l of hybridization mixture was added and incubated for 5 min in a water bath
at 60C. Finally, 300 l of selection reagent was added and incubated for 5 min
at 60C in a water bath. The results were measured with a luminometer (Leader;
Gen-Probe). A sample with a signal of equal to or more than 50,000 relative light
units was considered positive. With a signal of less than 50,000 relative light units,
the sample was considered negative.
(iv) Coagulase gene PCR. All discordant samples were also processed by PCR
using a specific primer set to detect the coagulase gene as published previously
(3). The PCR aims at the 3 end of the coagulase gene by employing primers
COAG2 and COAG3 (5-CGAGACCAAGATTCAACAAG-3 and 5-AAAG
AAAACCACTCACATCA-3, respectively). PCR was performed in a mixture of
10 mM Tris-HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl 2, 0.01% gelatin, 0.1%
Triton X-100, 0.2 mM (each) deoxynucleoside triphosphate, 50 pmol of the
respective primers, and 0.2 U of Tth polymerase (Supertaq; HT Biotechnology,
Cambridge, United Kingdom). The PCR machines were manufactured by
Biomed (Theres, Germany). No postamplification restriction analysis was performed;
the PCR was meant for detecting and not typing of the coagulase gene.
(v) Typing of S. aureus strains. To find out whether the discordant results were
caused by a single subset of clonally related strains, typing was performed for all
discordant strains and a random sample (n 10) of strains showing concordant
results. Typing was performed by arbitrarily primed PCR (AP-PCR) as described
previously (5, 7–9). The buffer conditions were as described above, and the
primers used were the same as those described in reference 5.
Table 1 shows the sensitivity of the various tests for methicillin-susceptible
S. aureus (MSSA) and on MRSA. The sensitivity
for MSSA was 100% for all but one test, Staphaurex
MSSA (n 222) MRSA (n 78) Total (n 300)
Staphaurex Plus 0 100 0 100 0 100
Staphaurex 2 99.1 12 84.6 14 95.3
Pastorex Staphplus 0 100 0 100 0 100
Free coagulase 0 100 6 92.3 6 98.0
Bound coagulase 0 100 3 96.1 3 99.0
TABLE 2. Diagnostic data obtained for discordant S. aureus strains
(sensitivity, 99.1%). However, for MRSA only Staphaurex Plus
and Pastorex Staphplus had a sensitivity of 100%. Again,
Staphaurex had the lowest sensitivity, 84.6%.
Out of 330 staphylococcal isolates tested, 30 gave negative
results in all tests and were considered not to be S. aureus. This
included results by Accuprobe and coagulase PCR testing.
Two hundred eighty gave positive results in all tests. The remaining
20 isolates (18 MRSA and 2 MSSA isolates [Table 2])
gave discordant results, meaning a negative result in one or two
of the five identification assays. Strains 7 to 20 were negative in
the Staphaurex assay, whereas strains 1 to 6 did not seem to
produce free coagulase. Three of the latter strains did not
produce bound coagulase as well. Interestingly, all strains appeared
to contain the (silenced?) coagulase gene as determined
by PCR, whereas also the Accuprobe assay identified
the strains as S. aureus. The 20 strains mentioned in Table 2
were derived from 11 patients. Repeated analysis performed
for these discordant isolates corroborated the initial data. The
additional tests for detection of free coagulase were also negative
on the six MRSA strains from Polish patients with negative
outcomes by the Difco coagulase plasma test. All 20
discordant isolates gave positive results in the Accuprobe assay
and the coagulase gene PCR. Therefore, these 20 isolates were
still considered to be S. aureus.
Test result a
1 1 3
2 2 3
3 3 3
4 4 3
5 5 3
6 6 3
7 7 2
8 7 2
9 7 2
10 7 2
11 7 2
12 7 2
13 7 2
14 8 2
15 8 2
16 8 2
17 8 2
18 9 2
19 10 4
20 11 1
a , positive; , negative.
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VOL. 34, 1996 COMPARISON OF TESTS FOR S. AUREUS IDENTIFICATION 2269
FIG. 1. Random amplification of polymorphic DNA analysis for the discordant
S. aureus strains. Strain numbers are indicated above the lanes, and the
positions of the molecular size markers (Pharmacia 100-bp ladder) are indicated
(lane mw). The upper panel displays the results of application of the primer
RAPD1; the lower panel summarizes the results of application of the primer
ERIC2. For assessing reproducibility, multiple isolates of strains 1 and 4 were
included. Strain numbering corresponds to that used in Table 2.
The results of AP-PCR revealed that the 20 discordant isolates
from 11 patients derived from only four clones (Fig. 1
[shows examples of two of the clones] and Table 2). Ten randomly
selected control strains showed 10 different patterns
(results not shown; see also references 5, 7, 8, and 9). This
proves the discriminatory power of the AP-PCR method used.
Pattern 1 was found for only one patient with discordant results,
pattern 2 was found for three patients involved in a small
outbreak of MRSA in our hospital, and pattern 3 was found for
six patients from Poland, probably involved in an outbreak of
MRSA. Pattern 4 was an MSSA strain.
Both Staphaurex Plus and Pastorex Staphplus showed an
optimal sensitivity for the identification of both MSSA and
MRSA. The initial format of Staphaurex had the lowest sensitivity,
especially with MRSA, as reported previously (2). A
remarkable finding was the failure of the tests to detect free
coagulase in six MRSA strains from Poland. Also, the test to
detect clumping factor was negative in three of these six
strains. This is worrisome, since detection of free coagulase is
considered to be the gold standard (4). It should be noted that
these six strains represented one clone. Both the PCR for the
coagulase gene and the Accuprobe test were positive with
these isolates. An explanation could be that the coagulase
gene, though present, is not expressed or is insufficiently expressed
in these strains. The specificity of all tests was 100%,
although the number of non-S. aureus isolates (total, 30) is
insufficient to draw definite conclusions with regard to the
absolute specificity. The value of genetic typing methods in this
kind of analysis is shown in Table 2. The 20 isolates with
discordant results represented only four clonally related
strains. In conclusion, Staphaurex Plus and Pastorex Staphplus
have excellent sensitivity for identifying S. aureus in clinical
samples, which makes them reliable screening tests in the clinical
This study was financially supported by Murex Diagnostics Limited.
1. Bauer, A. W., W. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic
susceptibility testing by a standardized single disk method. Am. J. Clin.
2. Fournier, J. M., A. Bouvet, D. Mathieu, F. Nato, A. Boutonnier, R. Gerbal,
P. Brunengo, C. Saulnier, N. Sagot, B. Slizewicz, and J. C. Mazie. 1993. New
latex reagent using monoclonal antibodies to capsular polysaccharide for
reliable identification of both oxacillin-susceptible and oxacillin-resistant
Staphylococcus aureus. J. Clin. Microbiol. 31:1342–1344.
3. Goh, S.-H., S. K. Byrne, J. L. Zhang, and A. W. Chow. 1992. Molecular typing
of Staphylococcus aureus on the basis of coagulase gene polymorphisms.
J. Clin. Microbiol. 30:1642–1645.
4. Kloos, W. E., and D. W. Lambe, Jr. 1991. Staphylococcus, p. 222–237. In A.
Balows, W. J. Hausler, Jr., K. L. Hermann, H. D. Isenberg, and H. J.
Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society
for Microbiology, Washington, D.C.
5. Kluytmans, J., W. van Leeuwen, W. Goessens, R. Hollis, S. Messer, L.
Herwaldt, H. Bruining, M. Heck, J. Rost, N. van Leeuwen, A. van Belkum,
and H. Verbrugh. 1995. Food-initiated outbreak of methicillin-resistant
Staphylococcus aureus analyzed by pheno- and genotyping. J. Clin. Microbiol.
6. National Committee for Clinical Laboratory Standards. 1990. Performance
standards for antimicrobial disk susceptibility tests, 4th ed. Approved standard
M2-A4, vol. 10. National Committee for Clinical Laboratory Standards,
7. van Belkum, A., R. Bax, P. Peerbooms, W. H. F. Goessens, N. van Leeuwen,
and W. G. V. Quint. 1993. Comparison of phage typing and DNA fingerprinting
by polymerase chain reaction for discriminating methicillin-resistant
Staphylococcus aureus strains. J. Clin. Microbiol. 31:798–803.
8. Van Belkum, A., R. Bax, and G. Prevost. 1994. Comparison of 4 genotyping
assays for epidemiological study of methicillin resistant S. aureus. Eur.
J. Clin. Microbiol. Infect. Dis. 13:420–424.
9. van Belkum, A., J. Kluytmans, W. van Leeuwen, R. Bax, W. Quint, E. Peters,
A. Fluit, C. Vandenbroucke-Grauls, A. van den Brule, H. Koeleman, W.
Melchers, J. Meis, A. Elaichouni, M. Vaneechoutte, F. Moonens, N. Maes,
M. Struelens, F. Tenover, and H. Verbrugh. 1995. Multicenter evaluation of
arbitrarily primed PCR for typing of Staphylococcus aureus strains. J. Clin.
10. Van der Vijver, J. C., C. A. Kraayeveld, and M. F. Michel. 1972. A solid
medium for visual demonstration of coagulase production by Staphylococcus
aureus. J. Clin. Pathol. 25:450–452.
jcm.highwire.org by on November 11, 2009