AM. Freydiere1, M. Bes2, MA. Charveriat3 , A. Carricajo3, C. Ploton1

1Hôpital Debrousse, Hospices Civils de Lyon, 2CNR Toxémies à Staphylocoques, Lyon, France, 3Hôpital de Bellevue, St Etienne France.

STAPHYCHROM II, A NEW SPECIFIC STAPHYLOCOAGULASE TEST USING A CHROMOGENIC SUBSTRATESTAPHYCHROM II, A NEW SPECIFIC STAPHYLOCOAGULASE TEST USING A CHROMOGENIC SUBSTRATEP 392 12th ECCMID Milan

24-27 April 2002e-mail: am.freydiere@chu-lyon.fr

IntroductionIntroduction

Results are summarised in Table 1 for the 113 clinical isolates and in Table 2 for the 15 collection strains.ResultsResults

ConclusionConclusion

ReferencesReferences

AbstractAbstract Objectives : Tested with 297 Staphylococcus strains, Staphychrom (International Microbio, France), a 2 hours fluorogenic test using human prothrombin and protease inhibitors for the detection of staphylocoagulase has been shown to be more specific, and as sensitive as the reference tube coagulase test (TCT), and more specific and more sensitive than a latex agglutination test (LAT). Staphychrom II, a new version of Staphychrom, uses a chromogenic substrate and a simplified methodology. This study compared Staphychrom II to (i) Staphychrom, (ii) a reference TCT using rabbit plasma + EDTA and (iii) the LAT Slidex Staph Plus (bioMérieux, France) for the rapid identification of Staphylococcus aureus.Methods: 113 clinical isolates including 92 S. aureus (78 methicillin sensitive and 14 methicillin resistant), and 21 S. non-aureus, were tested.Moreover,15 collection strains: 4 S. aureus (2 of which with a negative reference TCT) and 11 S. non-aureus, selected for their ability to give false-positive results either with the TCT or a LAT, were tested. Results : With the clinical isolates, Staphychrom II, Staphychrom and the TCT yielded a positive-coagulase test with 91 of 92 S. aureus strains (sensitivity 98.9%) then a negative coagulase-test with the 21 of 21 S. non-aureus (specificity 100 %). The LAT yielded a negative-agglutination with 18 of 21 S. non-aureus and 3 S. aureus strains (1 methicillin sensitive and 2 methicillin resistant) (sensitivity 96.7 % and specificity 85.7%).With the collection strains, Staphychrom II and Staphychrom yielded a positive-coagulase test with 2 of 4 S. aureus and a negative-coagulase test with the 11 S. non-aureus and the 2 S. aureus known to be negative with the reference TCT. The TCT yielded a positive test with 2 of 4 S. aureus and 8 of 11 S. non-aureus strains (3 S. schleiferi subsp coagulans and 4 S. intermedius and 1 S. delphini). The LAT yielded a positive-agglutination test with 3 of 4 S. aureus and 7 S. non-aureus strains (2 S. schleiferi subsp coagulans, 3 S. intermedius, 1 S. hyicus and 1 S. delphini).Conclusions : Staphychrom II yields the same results as Staphychrom. This test is as sensitive and more specific than the TCT, and more sensitive and more specific than the LAT Slidex Staph Plus.This rapid, accurate and easy-to-use coagulase test seems to be a good substitute for the conventional TCT.

Tested with 297 Staphylococcus strains, Staphychrom (International Microbio, France), a 2 hours fluorogenic test using human prothrombin and protease inhibitors for the detection of staphylocoagulase has been shown to be more specific, and as sensitive as the reference tube coagulase test (TCT), and more specific and more sensitive than the latex agglutination test (LAT) Pastorex Staph Plus (Bio-Rad, France) (1, 2).Objectives: The aim of this work is to comparatively assess the performance of Staphychrom II, a new version of Staphychrom, using a chromogenic substrate and a simplified methodology, with (i) Staphychrom, (ii) a reference TCT and (iii) the LAT Slidex Staph Plus (bioMérieux, France) for the rapid identification of Staphylococcus aureus.

Material and MethodsMaterial and MethodsStrains: - 113 fresh isolates from inpatients from 2 hospitals including 92 S. aureus and 21 S. non-aureus strains were tested.These isolates were isolated from various specimens (57 pus , 27 nose, 13 broncho alveolar fluid, 8 stool, 3 catheter, 3 throat, and 2 blood specimens).- 15 reference isolates including 4 S. aureus (2 of which with a negative reference TCT) and 11 S. non-aureus, selected for their ability to give false-positive results either with the TCT or a LAT, were tested. Reagents and methodology:Slidex Staph Plus (bioMérieux, France) is a latex agglutination test (LAT) that simultaneously detects clumping factor, protein A, and specific S. aureus capsular polysaccharides. This test was used according to the manufacturer’s recommendations.Conventional tube coagulase test (3) For this test, strains were incubated an overnight at 37°C in a staphylocoagulase broth (BioRad, France). A 0.05 ml volume of this broth were added to 0.5 ml of Rabbit plasma containing EDTA (Becton Dickinson, France) and mixed. Tests were incubated at 37°C and examined at 4 and 24 h.Staphychrom (International Microbio, France) is a 2 hours staphylocoagulase test using human prothrombin and protease inhibitors (4). Staphychrom II, a new version of Staphychrom uses a chromogenic substrate instead of a fluorogenic substrate for Staphychrom. The two tests are inoculated as indicated in Figure 1. Interpretation: When a discrepant result between the Staphychrom test or the Slidex Staph Plus or/and the conventional coagulase test happened, staphylococci were identified by the ID32 STAPH system (bioMérieux, France) and/or the Accuprobe S. aureus test (bioMérieux, France) which detects the specific rRNA sequences unique to S. aureus.

1. Treny A, M. Bes, N. Fonsale, A. Carricajo, M.E. Reverdy and A.M. Freydiere. 2001. Staphychrom, a new specific staphylocoagulase test using a fluorogenic substrate abstr. P1512. In Abstract 11th European Congress of Clinical Microbiology and Infectious Diseases. Istanbul, Turkey, 1-4 April. Clin. Microbiol. Infect. 2001, 7 (suppl. 1): 326-327.2. Carricajo A, A. Treny, N. Fonsale, M. Bes, M.E. Reverdy, Y. Gille, G. Aubert and A.M. Freydiere. 2001. Performance of the Chromogenic Medium CHROMagar Staph aureus and the Staphychrom Coagulase Test in the Detection and the Identification of Staphylococcus aureus in Clinical Specimens. J. Clin. Microbiol. 39: 2581-2583.3. Kloos W.E. and T.L. Bannerman. 1995. Staphylococcus and Micrococcus, p. 282-298. In P. R. Murray, E. J. Baron, M. A. Pfaller, F.C. Tenover, and R. H. Yolken (ed), Manual of Clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.4. Langlet S., G. Quentin, G. Contant and J.C. Ghnassia. 1999. Méthode chromogénique d’identification rapide de Staphylococcus aureus. Ann. Biol. Clin. 57: 191-196.5. Célard M., F. Vandenesch, H. Darbas, J. Grando, H. Jean-Pierre, G. Kirkorian and J. Etienne. 1997. Pacemaker infection caused by Staphylococcus schleiferi, a member of the human preaxillary flora: four case reports. Clin. Infect. Dis. 24: 1014-1015.6. Leung M.J., N. Nuttall, M. Mazur, T.L. Taddei, M. McComish and J.W. Pearman. 1999. Case of Staphylococcus schleiferi endocarditis and a simple scheme to identify clumping factor-positive staphylococci. J. Clin. Microbiol. 37: 3353-3356.7. Mahoudeau I., X. Delabranche, G. Prevost, H. Monteil and Y. Piemont. 1997. Frequency of isolation of Staphylococcus intermedius from humans. J. Clin. Microbiol. 35: 2153-2154.8. Vandenesch F., M. Célard, D. Arpin, M. Bes, T. Greenland and J. Etienne. 1995. Catheter-related bacteremia associated with coagulase-positive Staphylococcus intermedius. J. Clin. Microbiol. 33: 2508-2510.9. Kluytmans J, H. Berg, P. Steegh, F. Vandenesch, J. Etienne and A. van Belkum. 1998. Outbreak of Staphylococcus schleiferi wound infections: strain characterization by randomly amplified polymorphic DNA analysis, PCR ribotyping, conventional ribotyping, and pulsed-field gel electrophoresis. J. Clin Microbiol. 36: 2214-2219.

Like Staphychrom previously tested with 297 Staphylococcus strains (1, 2), Staphychrom II has been shown to be :

more specific, more rapid and as sensitive as the reference tube coagulase test, more specific and more sensitive but less rapid than the latex agglutination tests

This rapid, accurate and easy-to-use coagulase test is a good substitute for the conventional TCT and additional testings are being performed to determine if it could be used as an identification test avoiding the frequently recommended combination of two tests for accurate identification of S. aureus.

With the 113 clinical isolates, both Staphychrom II, Staphychrom and the conventional coagulase test showed a sensitivity and a specificity of 98.9% and 100 %, respectively, while the Slidex Staph Plus LAT yielded a sensitivity and a specificity of 96.7 % and 85.7%, respectively.These results of sensitivity and specificity of Staphychrom II using a simplified methodology are in complete agreement with those obtained with previous work (1, 2). In contrast, Slidex Staph Plus yielded a negative agglutination test with 3 S. aureus strains, 2 of which being MRSA.

With the 2 S. aureus strains selected as being coagulase-negative by different methods, Staphychrom II and Staphychrom, gave a negative result. In contrast, the Slidex Staph Plus LAT gave a positive result with one of these two strains. With the 15 strains selected for their ability to give false-positive results either with the TCT or a LAT, Staphychrom II and Staphychrom yielded a specificity of 100 %.Although species (e.g. S. schleiferi, S. intermedius) giving non specific reaction either with the conventional coagulase test or with the Slidex Plus test, are not commonly encountered in clinical samples, several authors (5-9) have underlined the necessity to accurately identify these species in some clinical situations.

Acknoledgements.Acknoledgements. We thank our respective technicians for their assistance, International Microbio for providing us with the reagents and A. Frangin for her valuable help in producing this poster.

Figure 1

The tray : 8 wells containing a culture medium adaptated for staphylococci and a specific chromogenic substrate of staphylocoagulase

Pick up one isolated colony with the inoculator PRESTO ABG®

2

Mix the colony in the Test well. Repeat twice the inoculation

with the same PRESTO ABG®.

Test wellControl well1 to 2 hours incubation

at 37°C

3

4

Reading can only be interpreted if the control well shows no yellow coloration.

Yellow coloration S. aureus

Test wellControl well

No coloration Negative

5

Coag reagent: Lyophylized medium containing purified human prothrombin and NaCl for 2 ml sterile distilled water

2 drops of Coag reagent

in 2 wells

1

Table 1. Comparaison of Staphychrom II to (i) Staphychrom (ii) a conventional coagulase test and the latex agglutina-tion test Slidex Staph Plus by using 113 clinical isolates.

a : 14 strains corresponded to Methicillin Resistant Staphylococcus aureus strains, b : 2 strains corresponded to Methicillin Resistant Staphylococcus aureus strains

S. species Tested

strains

Staphychrom II

chromogenic

Staphychrom

fluorogenic

Conventional

coagulase test

Slidex Staph

Plus

+ - + - + - + -

S. aureus 92a 91 1 91 1 91 1 89 3 b

S. non-aureus 21 0 21 0 21 0 21 3 18

a : 2 of the 4 S. aureus strains yielded a negative reference TCT, b : 3 S. schleiferi subsp coagulans and 4 S. intermedius and 1 S. delphini.c : 2 S. schleiferi subsp coagulans, 3 S. intermedius, 1 S. hyicus and 1 S. delphini

Table 2. Comparaison of Staphychrom II to (i) Staphychrom (ii) a conventional coagulase test and the latex agglutination test Slidex Staph Plus by using 15 reference strains.

S. species Tested

strains

Staphychrom II

chromogenic

Staphychrom

fluorogenic

Conventional

coagulase test

Slidex

Staph Plus

+ - + - + - + -

S. aureus 4 a 2 2 2 2 2 2 3 1

S. non-aureus 11 0 11 0 11 8 b 3 7 c 4