Staphylococcus aureus

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Staphylococcus aureus

1. 검 사 원 리 - 라텍스 응집법 (Latex-Agglutination Method)

- 1 -

S.aureus(latex 응집법)

- 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

음성으로 보고한다.

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

텍스 시약

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

1) 보고방법

- 음성대조액의 응집이 없어야 한다.

- 2 -

S.aureus(latex 응집법)

- 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) 내부정도관리

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

0095-1137/96/$04.000

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.

2267

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

included.

(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

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2268 LUIJENDIJK ET AL. J. CLIN. MICROBIOL.

Test

TABLE 1. Survey of the results of the different S. aureus identification assays

No. of

false-negative

results

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.

RESULTS

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)

Test

sensitivity

(%)

No. of

false-negative

results

Test

sensitivity

(%)

No. of

false-negative

results

Test

sensitivity

(%)

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

Strain

no.

Patient

no.

Staphaurex

Plus

TABLE 2. Diagnostic data obtained for discordant S. aureus strains

Staphaurex

Pastorex

Staphplus

(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

Free

coagulase

Bound

coagulase

Accuprobe

PCR for

coagulase gene

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.

AP-PCR

pattern

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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.

DISCUSSION

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

microbiology laboratory.

ACKNOWLEDGMENT

This study was financially supported by Murex Diagnostics Limited.

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