Journal of Clinical Pathology, 1979, 32, 1234-1236
Grouping of streptococci by Streptex SHEENA A. WAITKINS, J. G. RATCLIFFE, R. D. ANDERSON, AND C. ROBERTS From the Regional Public Health Laboratory, Fazakerley Hospital, Lower Lane, Liverpool L9 7AL, UK
Streptex was compared to routine laboratory identification methods available. The results from Streptex sometimes required several attempts before final identification could be achieved. In the main, group D streptococci other than Strep. faecalis failed to group with the Streptex antisera, and this method cannot therefore be used exclusively as a means of identifying this group of streptococci.
SUMMARY
The identification of /-haemolytic streptococci is very time-consuming and technically laborious, particularly when the classical Lancefield grouping methods are used (Fuller, 1938; Lancefield, 1938). An improved rapid method of Lancefield grouping was developed by Rosendal (1956) using a slide agglutination of trypsinised streptococcal suspension. This was further modified by Christensen et al. (1973) and Maxted et al. (1976) to incorporate the staphylococcal protein A coagglutination technique, which results in instant flocculation of the trypsinised streptococcal suspension. These methods depend on a supply of protein A and the homologous sera. Individual small-scale production of antisera for coagglutination methods would not be possible in a busy routine clinical laboratory. Recently, Wellcome Laboratories have modified this coagglutination system by substituting polystyrene latex particles for staphylococcal protein A and using the enzyme pronase for the antigen extraction (Ederer et al., 1972). This has been launched commercially as Streptex and is marketed for the identification of Lancefield groups A, B, C, D, F, and G. We report our experience using Streptex and its reliability and performance when compared with our own established laboratory routine.
inoculated and incubated anaerobically at 370C. The next morning the type and degree of pigmentation (Islam's plate), the haemolysis, and bacitracin sensitivity were recorded. If the test organism produced f-haemolysis, was bacitracin-resistant, and pigmented it was provisionally identified as group B, to be confirmed by coagglutination as described by Maxted et al. (1976). A non-pigmented streptococcus (ie, Islam's negative), which failed to group with A, B, C, or G antisera (supplied by the Division of Hospital Infection, Colindale), was further examined for pyruvate fermentation (WaitPrimary plate pure, isolated ccdonies- BacitracirS} record
haemolysisT"rcr
13 or hazy f3-hoem colony
Bacitracin Resistant
Sensitive
Islams Agar
Co-agglutination} record Colindale method A,B,C,orG
+
ve
-ve
Method ate fermentation
Well-isolated colonies on blood agar were replated onto 5% horse blood agar with a bacitracin disc (01 unit) and incubated aerobically at 370C. At the same time an Islam's plate (Islam, 1977) was Received for publication 2 July 1979
group B
I+
+ve
Strep faecalis
-ve Full biochemical identification
Figure Protocol for routine streptococcal identification methods. 1234
1235
Grouping of streptococci by Streptex
kins, 1978). If the latter was positive the organism was presumptively identified as Streptococcus faecalis, whereas if pyruvate fermentation was negative then further biochemical identification as described by Colman and Williams (1972) was undertaken. The flow chart (Figure) of streptococcal identification was adopted for the rapid routine identification of clinical isolates in our laboratory and directly compared with Streptex using the methods recommended by the manufacturers. Results
Of the 30 strains investigated (Table), 20 were routine clinical isolates from a variety of specimens, while the remaining 10 were reference strains. In all cases where positive identification was achieved with the Colindale method this was always at the first attempt, whereas in six instances Streptex gave
an
initial negative result and only repetition of the
test established the identification. Since
group
D
antiserum is absent from the Colindale reagents, identification of these organisms had to be based on biochemical methods. The Streptex performed poorly for those group D streptococci other than Strep. faecalis, and in one instance R75/1603 (Strep. faecalis serotype 19) failed to group at all. With other isolates (79/5911 and 79/6854), identified as groups B and C respectively, there appeared to be weak but detectable agglutination with other Streptex antisera. In general, Streptex reactions were not always easily readable, and identification was based on the strongest agglutination noted. Discussion We have examined only 30 streptococcal strains on this comparative basis, and although the Colindale
Table Comparison of Streptex with routine streptococcal identification methods Number
Haemolysis
Bacitracin
Islam's plate
Coagglutination -
R/7174 R/86264 R75/1065
, a
R/8174 78/91066 79/1998 79/1295 79/2296 79/2836 79/5911 79/5620 79/5946 79/5843 79/6017 79/6746 79/6564 79/6854 79/8611 78/84599 R75/1096 R75/1886 R75/1611 R75/1601 R75/1615 R75/1603 79/13010
79/5620 R/7366 R/10231 R/7864
, ,8
/3 is ,B
is ,
is ,
9 is is 9 a a
is O ,B
R R R R R S R R S R R R R R S S R S R R R R R R R R
-
+±++ -
-
++++ ++++ ++++ _ ++++ ++++ -
+++ -
-
+++
9
WS
-
is is
S S S
-
Colindale method
Streptex
NA B NA NA
B D (x2) (weak) D (+++) A(x2) B B A(x3) B (x 3)*
NA A B B A B G B C B G A C A B NA NA NA NA NA NA B G _ _
G(x3) B (x 3) C B (weak) G A C** A B D (weak) D D B G _ _
R = resistant; S = sensitive; WS = weak sensitive NA = not available; ND = not done ( x -) = number of times Streptex was repeated before a positive agglutination
Results: + - + + + + degrees of positive - negative Notes:
*Weak detectable reaction with G
**
Weak detectable reaction with B and G
Pyruvate fermentation
Biochemical identification
Strain identified
ND _ -
Yes ND Yes Yes
Strep. faecium Group B Strep. faecium Strep. durans
+++ ND ND ND ND
Yes ND ND ND ND ND ND ND ND ND ND ND ND ND ND Yes Yes Yes Yes Yes Yes ND ND Yes Yes Yes
Strep. faecalis 1 Group A Group B Group B Group A Group B Group G Group B Group C Group B
ND ND ND ND ND ND ND ND ND ND +++ +++ +++ ++ ND ND _ _
Group G Group A Group C Group A Group B Strep. bovis I Strep. bovis *I Strep. faecalis 5 Strep. faecalis I Strep. faecalis 9 Strep. faecalis 19 Group B Group G Group K Strep. sanguis H Strep. sanguis
1236
Sheena A. Waitkins, J. G. Ratcliffe, R. D. Anderson, and C. Roberts
coagglutination antiserum readily identifies strep- Colman, G., and Williams, R. E. 0. (1972). Taxonomy of some human viridans streptozocci. In: Streptococci tococci of groups A, B, C, and G, its disadvantage is and Streptococcal Diseases: Recognition, Understandthe limited availability of thereagentsandtheabsence ing and Management. Edited by L. W. Wannamaker of a group D antiserum. There is no doubt that if and J. M. Mats-n. New York, London. Streptex is the only coagglutination method available Christensen, P., Kahlmeter, G., Jonsson, S., and Kronvall, it is a considerable advance over other methods and G. (1973). 'New method for the serological grouping provides an additional useful tool for the identiof streptococci with specific antibodies adsorbed to fication of streptococci, particularly non-group D protein A-containing staphylococci'. Infection and Immunity, 7, 881-885. isolates. However, in our hands, some of the typing reactions were weak and required repetition, while Ederer, G. M., Herrmann, M. M., Bruce, R., Masten, J, M., and Chapman, S. S. (1972). 'Rapid extraction only five of the 10 group D streptococci tested were method with pronase B for grouping beta haemolytic grouped satisfactorily. Of these, four were Strep. streptococci'. Applied Microbiology, 23, 285-288. faecalis and one was Strep. durans. Tlhe five ungroup- Fuller, A. T. (1938). 'The formamide method for the able strains included two cultures each of Strep. extraction of polysaccharides from haemolytic strepfaecium and Strep. bovis. Tht clinical relevance of tococci'. British Journal of Experimental Pathology, 19, the latter is that endocarditis due to Strep. bovis 130-139. appears to be restricted to the middle-aged and Islam, A. K. M. S. (1977). 'Rapid recognition of group B streptococci' (Letter). Lancet, 1, 256-257. elderly, particularly in association with carcinoma Lancefield, R. C. (1938). 'A microprecipitin-technic for of the colon (Ahmed and Meerhan, 1978). classifying hemolytic streptococci, and improved Although this is contrary to the Streptex method, methods for producing antisera'. Proceedings of the we feel it is necessary to employ pure cultures. The Society for Experimental Biology and Medicine, 38, attempted identification of a suspected group A 473-478. streptococcus from a plate with mixed normal flora Maxted, W. R., Efstratiou, A., and Parker, M. T. (1976). may compromise the identification of that organism. 'Agglutination grouping of streptococci' (Letter). This is because certain 'viridans' streptococci Lancet, 2, 692-693. possess Lancefield group antigens, therefore yielding Rosendal, K. (1956). 'Grouping of hemolytic streptococci belonging to groups A, C, and G. Acta diverse and complicated coagglutination results. Pathologica et Microbiologica Scandinavica, 39, In conclusion, we find the Streptex a useful 127-136. laboratory tool provided that the above criticisms Waitkins, S. A. (1978). 'Use of pyruvate fermentation are borne in mind when interpreting the results. compared with tetrazolium reduction in the differentiation of group D streptococci'. Journal of Clinical We thank Wellcome Ltd for the supply of Streptex. Pathology, 31, 692-695.
References Ahmad, S., and Meerhan, M. E. (1978). Streptococcus bovis endocarditis with carcinoma of the colon (Letter). British Medical Journal, 2, 1166.
Requests for reprints to: Dr S. A. Waitkins, Regional Public Health Laboratory, Fazakerley Hospital, Lower Lane, Liverpool L9 7AL, UK.
Grouping of streptococci by Streptex SHEENA A. WAITKINS, J. G. RATCLIFFE, R. D. ANDERSON, AND C. ROBERTS From the Regional Public Health Laboratory, Fazakerley Hospital, Lower Lane, Liverpool L9 7AL, UK
Streptex was compared to routine laboratory identification methods available. The results from Streptex sometimes required several attempts before final identification could be achieved. In the main, group D streptococci other than Strep. faecalis failed to group with the Streptex antisera, and this method cannot therefore be used exclusively as a means of identifying this group of streptococci.
SUMMARY
The identification of /-haemolytic streptococci is very time-consuming and technically laborious, particularly when the classical Lancefield grouping methods are used (Fuller, 1938; Lancefield, 1938). An improved rapid method of Lancefield grouping was developed by Rosendal (1956) using a slide agglutination of trypsinised streptococcal suspension. This was further modified by Christensen et al. (1973) and Maxted et al. (1976) to incorporate the staphylococcal protein A coagglutination technique, which results in instant flocculation of the trypsinised streptococcal suspension. These methods depend on a supply of protein A and the homologous sera. Individual small-scale production of antisera for coagglutination methods would not be possible in a busy routine clinical laboratory. Recently, Wellcome Laboratories have modified this coagglutination system by substituting polystyrene latex particles for staphylococcal protein A and using the enzyme pronase for the antigen extraction (Ederer et al., 1972). This has been launched commercially as Streptex and is marketed for the identification of Lancefield groups A, B, C, D, F, and G. We report our experience using Streptex and its reliability and performance when compared with our own established laboratory routine.
inoculated and incubated anaerobically at 370C. The next morning the type and degree of pigmentation (Islam's plate), the haemolysis, and bacitracin sensitivity were recorded. If the test organism produced f-haemolysis, was bacitracin-resistant, and pigmented it was provisionally identified as group B, to be confirmed by coagglutination as described by Maxted et al. (1976). A non-pigmented streptococcus (ie, Islam's negative), which failed to group with A, B, C, or G antisera (supplied by the Division of Hospital Infection, Colindale), was further examined for pyruvate fermentation (WaitPrimary plate pure, isolated ccdonies- BacitracirS} record
haemolysisT"rcr
13 or hazy f3-hoem colony
Bacitracin Resistant
Sensitive
Islams Agar
Co-agglutination} record Colindale method A,B,C,orG
+
ve
-ve
Method ate fermentation
Well-isolated colonies on blood agar were replated onto 5% horse blood agar with a bacitracin disc (01 unit) and incubated aerobically at 370C. At the same time an Islam's plate (Islam, 1977) was Received for publication 2 July 1979
group B
I+
+ve
Strep faecalis
-ve Full biochemical identification
Figure Protocol for routine streptococcal identification methods. 1234
1235
Grouping of streptococci by Streptex
kins, 1978). If the latter was positive the organism was presumptively identified as Streptococcus faecalis, whereas if pyruvate fermentation was negative then further biochemical identification as described by Colman and Williams (1972) was undertaken. The flow chart (Figure) of streptococcal identification was adopted for the rapid routine identification of clinical isolates in our laboratory and directly compared with Streptex using the methods recommended by the manufacturers. Results
Of the 30 strains investigated (Table), 20 were routine clinical isolates from a variety of specimens, while the remaining 10 were reference strains. In all cases where positive identification was achieved with the Colindale method this was always at the first attempt, whereas in six instances Streptex gave
an
initial negative result and only repetition of the
test established the identification. Since
group
D
antiserum is absent from the Colindale reagents, identification of these organisms had to be based on biochemical methods. The Streptex performed poorly for those group D streptococci other than Strep. faecalis, and in one instance R75/1603 (Strep. faecalis serotype 19) failed to group at all. With other isolates (79/5911 and 79/6854), identified as groups B and C respectively, there appeared to be weak but detectable agglutination with other Streptex antisera. In general, Streptex reactions were not always easily readable, and identification was based on the strongest agglutination noted. Discussion We have examined only 30 streptococcal strains on this comparative basis, and although the Colindale
Table Comparison of Streptex with routine streptococcal identification methods Number
Haemolysis
Bacitracin
Islam's plate
Coagglutination -
R/7174 R/86264 R75/1065
, a
R/8174 78/91066 79/1998 79/1295 79/2296 79/2836 79/5911 79/5620 79/5946 79/5843 79/6017 79/6746 79/6564 79/6854 79/8611 78/84599 R75/1096 R75/1886 R75/1611 R75/1601 R75/1615 R75/1603 79/13010
79/5620 R/7366 R/10231 R/7864
, ,8
/3 is ,B
is ,
is ,
9 is is 9 a a
is O ,B
R R R R R S R R S R R R R R S S R S R R R R R R R R
-
+±++ -
-
++++ ++++ ++++ _ ++++ ++++ -
+++ -
-
+++
9
WS
-
is is
S S S
-
Colindale method
Streptex
NA B NA NA
B D (x2) (weak) D (+++) A(x2) B B A(x3) B (x 3)*
NA A B B A B G B C B G A C A B NA NA NA NA NA NA B G _ _
G(x3) B (x 3) C B (weak) G A C** A B D (weak) D D B G _ _
R = resistant; S = sensitive; WS = weak sensitive NA = not available; ND = not done ( x -) = number of times Streptex was repeated before a positive agglutination
Results: + - + + + + degrees of positive - negative Notes:
*Weak detectable reaction with G
**
Weak detectable reaction with B and G
Pyruvate fermentation
Biochemical identification
Strain identified
ND _ -
Yes ND Yes Yes
Strep. faecium Group B Strep. faecium Strep. durans
+++ ND ND ND ND
Yes ND ND ND ND ND ND ND ND ND ND ND ND ND ND Yes Yes Yes Yes Yes Yes ND ND Yes Yes Yes
Strep. faecalis 1 Group A Group B Group B Group A Group B Group G Group B Group C Group B
ND ND ND ND ND ND ND ND ND ND +++ +++ +++ ++ ND ND _ _
Group G Group A Group C Group A Group B Strep. bovis I Strep. bovis *I Strep. faecalis 5 Strep. faecalis I Strep. faecalis 9 Strep. faecalis 19 Group B Group G Group K Strep. sanguis H Strep. sanguis
1236
Sheena A. Waitkins, J. G. Ratcliffe, R. D. Anderson, and C. Roberts
coagglutination antiserum readily identifies strep- Colman, G., and Williams, R. E. 0. (1972). Taxonomy of some human viridans streptozocci. In: Streptococci tococci of groups A, B, C, and G, its disadvantage is and Streptococcal Diseases: Recognition, Understandthe limited availability of thereagentsandtheabsence ing and Management. Edited by L. W. Wannamaker of a group D antiserum. There is no doubt that if and J. M. Mats-n. New York, London. Streptex is the only coagglutination method available Christensen, P., Kahlmeter, G., Jonsson, S., and Kronvall, it is a considerable advance over other methods and G. (1973). 'New method for the serological grouping provides an additional useful tool for the identiof streptococci with specific antibodies adsorbed to fication of streptococci, particularly non-group D protein A-containing staphylococci'. Infection and Immunity, 7, 881-885. isolates. However, in our hands, some of the typing reactions were weak and required repetition, while Ederer, G. M., Herrmann, M. M., Bruce, R., Masten, J, M., and Chapman, S. S. (1972). 'Rapid extraction only five of the 10 group D streptococci tested were method with pronase B for grouping beta haemolytic grouped satisfactorily. Of these, four were Strep. streptococci'. Applied Microbiology, 23, 285-288. faecalis and one was Strep. durans. Tlhe five ungroup- Fuller, A. T. (1938). 'The formamide method for the able strains included two cultures each of Strep. extraction of polysaccharides from haemolytic strepfaecium and Strep. bovis. Tht clinical relevance of tococci'. British Journal of Experimental Pathology, 19, the latter is that endocarditis due to Strep. bovis 130-139. appears to be restricted to the middle-aged and Islam, A. K. M. S. (1977). 'Rapid recognition of group B streptococci' (Letter). Lancet, 1, 256-257. elderly, particularly in association with carcinoma Lancefield, R. C. (1938). 'A microprecipitin-technic for of the colon (Ahmed and Meerhan, 1978). classifying hemolytic streptococci, and improved Although this is contrary to the Streptex method, methods for producing antisera'. Proceedings of the we feel it is necessary to employ pure cultures. The Society for Experimental Biology and Medicine, 38, attempted identification of a suspected group A 473-478. streptococcus from a plate with mixed normal flora Maxted, W. R., Efstratiou, A., and Parker, M. T. (1976). may compromise the identification of that organism. 'Agglutination grouping of streptococci' (Letter). This is because certain 'viridans' streptococci Lancet, 2, 692-693. possess Lancefield group antigens, therefore yielding Rosendal, K. (1956). 'Grouping of hemolytic streptococci belonging to groups A, C, and G. Acta diverse and complicated coagglutination results. Pathologica et Microbiologica Scandinavica, 39, In conclusion, we find the Streptex a useful 127-136. laboratory tool provided that the above criticisms Waitkins, S. A. (1978). 'Use of pyruvate fermentation are borne in mind when interpreting the results. compared with tetrazolium reduction in the differentiation of group D streptococci'. Journal of Clinical We thank Wellcome Ltd for the supply of Streptex. Pathology, 31, 692-695.
References Ahmad, S., and Meerhan, M. E. (1978). Streptococcus bovis endocarditis with carcinoma of the colon (Letter). British Medical Journal, 2, 1166.
Requests for reprints to: Dr S. A. Waitkins, Regional Public Health Laboratory, Fazakerley Hospital, Lower Lane, Liverpool L9 7AL, UK.
