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Serodiagnosis of staphylococcal infections
Leading Articles
Serodiagnosis of staphylococcal infections The usefulness of serological assays in the diagnosis of certain forms of staphylococcal infection has proved to be of some value when a staphylococcal aetiology is suspected but cannot be verified by culture. Measurement of neutralising antibody to the haemolytic activity of staphylococcal a-haemolysin was amongst the earliest methods chosen and for many years was the only method available’. However its applicability was dependent on a stable standardised preparation of the toxin which had been purified to improve its specificity to react with antibody. Being biological in nature, the assay displayed considerable inter-laboratory variation both in its specificity. sensitivity and interpretation. Similar assays which followed, detecting antibody to DNAase. beta-toxin, leucocidin and delta-toxin, suffered from the same criticisms’. Major developments in our knowledge of the structure of the bacterial cell wall, and of the Sraphvlococcus cell wall in particular, have led to the use of two components as antigens for the serological detection of staphylococcal disease’. Peptidoglycan (PG) is the major cell wall component representing about 5&60% of its dry weight. This component forms the basic matrix onto which other substances such as teichoic acid (TA) and protein A are covalently linked. However, only the latter two components are species-specific, whereas PC is present in virtually all procaryotic micro-organisms, although there are differences in fine detail between different species. Teichoic acid in particular has proved useful as an antigen in the detection of antibodies in cases of endocarditis, septicaemia and osteomyelitis caused by Stuphylococcus uureus. Numerous reports exist citing gel diffusion, counter-current immunoelectrophoresis (CIE) radioimmunoassay (RIA) and enzyme-linked immunosorbant assay (ELISA) as methods of choice to detect such antibodies. Raised levels of TA antibodies in endocarditis and septicaemia are a consistent feature of these reports, and it has been suggested that the TA can discriminate between endocarditis and other S. uureus infections, as only sera from S. aureus endocarditis had titres in excess of 4. With a commercially available TA antibody assay4 low levels of antibody were seen in 20% of normal individuals. Tines greater than 4 were considered positive and were regularly
seen in patients with uncomplicated bacteraemia. Despite the many favourable reports 01 TA detection by gel diffusion techniques, certain problems have limited the broader USCof these assays. Various technical aspects of the assays must be carefully controlled to minimise interlaboratory variability and intralaboratory reproducibility’. The sensitivity of the assay in patients with S. crureus endocarditis ranges from 61 to 100%. while the false positive rate in control subjects ranges from zero to 25%. Agar composition and the tcichoic acid content of the antigen used are two critical factors that can influence both the sensitivity and specificity of the assay. One diagnostic problem has been partially solved by the use of methods to detect teichoic acid antibodies. Hitherto. laboratory detection of septic arthritis has been dependent on Gram films, bacteriological culture and leucocyte counts, with varying amounts of success (3550%). However teichoic acid antibodies found by either gel diffusion or counter-current immunoelectrophoresis in either serum or synovial fluid have been successful. Of IO patients with clinical evidence of septic arthritis, four were shown to carry detectable levels of TAA by gel diffusion and another three patients were detected by CIE6. Synovial fluid was not such a suitable specimen in which to look for the antibody. It is likely that any antibody which is present is trapped in immune complexes as a consequence of antigen excess early in the infectious process. In this connection, it has been shown that staphylococcal antigens are present both free and complexed with antibody in various staphylococcal infections (endocarditis, bacteraemia and non-bacteraemic infections such as osteomyelitis). Circulating immune complexes were found in seven of eight patients with staphylococcal bacteraemia’. Thus antigen detection as well as TAA measurements may prove to be the most valuable in distinguishing patients with endocarditis from controls. Another test which may add to the quality of the laboratory detection of anti-staphylococcal antibodies is the parallel measurement of anti alpha-toxin antibodiesa. In an ELISA for antibodies to this toxin and teichoic acid, a 100% positive response was seen in 19 patients with paired serum samples from cases of endocarditis and complicated septicaemia. whilst an 89% rate was detected in uncomplicated bacteraermia. In this way, serological diagnosis of staphylococcal septicaemia and endocarditis
83
Leading Articles could be established in at least 97%“. Antibodies to a-haemolysin are positive in 65% of cases of staphylococcal osteomyelitis. Subsequent application of a test to measure antibody to a-haemolysin increased the detection rate in osteomyelitis to 80%1°. Anti-nuclease has also been tried in this connection giving slightly over 50% positivity in cases of staphylococcal osteomyelitis. Another approach which has been recently evaluated has been immunoblotting (also known as western blotting). By this means the patient’s antibody response to a variety of bacterial antigens can be assessed at one and the same time. Such a bacterial mosaic of antigens is prepared by subjecting whole-cell lysates to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferring them onto nitrocellulose (electroblotting). Any reaction with antibodies present in the patient’s serum can be visualised by an enzyme-linked immunosorbant assay. A study of sera obtained from individuals with osteomyelitis revealed the presence of an antigen of molecular weight 3 1,000 kDa which reacted with IgM antibody. Little or no antibody to this antigen was present in normal human sera”. A similar situation involving coagulasenegative staphylococci as the cause of prosthetic valve endocarditis has not until recently seen any developments in terms of serological measurements of specific antibodies. The clinical significance of a positive blood culture with coagulase-negative staphylococci in patients with prosthetic heart valves and fever can be difficult to determine. A serological assay capable of discriminating between endocarditis and uncomplicated bacteraemia is important since the form of therapy depends upon the in the former long-term antibiotic distinction therapy and/or valve replacement, in the latter short-term antibiotic therapy only. A solid-phase RIA has been developed’? with an ultrasonic cell extract which succeeded in this respect but failed to distinguish between cases of uncomplicated bacteraemia and mere contamination of blood culture bottles with coagulase-negative staphylococci. Later the same group” compared different antigens of S. t~pidertnidis (ultrasonic extract, purified cell walls, peptidoglycan and teichoic acid) for their specificity in diagnosing coagulase-negative staphylococcal endocarditis. However, the purified antigens were no better (or worse) than the ultrasonic extract used previously. This extract
contained at least 43 different antigensId yet was more specific than the purified antigens in an RIA. A quite different approach has been the detection of specific antibodies in patients on chronic ambulatory peritoneal dialysis (CAPD) who develop peritonitis”. An ELlSA with cellwall polysaccharide B as antigen was used such that in seven of I5 peritonitis episodes a rising titre of IgG antibody was measured. No such rise was seen in peritonitis caused by other bacteria. It would then appear that, at least in some instances, CAPD peritonitis due to coagulase-negative staphylococci results in a me;tsurable antibody response. However. even today the serodiagnosis of staphylococcal infections remains a minefield. represen!ed by the variety of antigens that have been used in the detection of specific antigens and the “sappers” who have developed more. and more sophisticated. methods of detection. The frequency with which we are exposed to staphylococci as antigenic stimuli blurs the threshold at which one can distinguish between commensal association and true infection.
References Mudd S. Gladstone GP, Lenhart NA, Hochstcin HD. Titrations of antibodies against alpha-haemolysin and the components of staphylococcal leucocidin in human subjects following immunization. Br J Exp Path 1962; 43: 313-9.
Taylor AG, Fincham WJ. Cook J. Staphylococcal antibodies
in osteomyelitis:
the use ofantisla-
phylococcal nuclease levels in diagnosis. In: Jeljaszewicz J, ed. Staphylococcal infections. Stuttgart:
G. Fischer,
1976: pp 9114.
Oeding P. Natas OB. Fleurette J. Detection of staphylococcal antibodies in human sera. In: MacDonald
A. Smith
G. eds. The staohvlococci.
Aberdeen: Aberdeen University Press: $7 108. Wheat J, Kohler RB, Garten M, White A. Commercially
available
(Endo-Staph)
assay for
tei-
choic acid antibodies. Evaluation in patients with serious Staphylococcus uureuS infections and in controls.
Arch
Intern
Med
1984; 144: 261 4.
Sheagrcn JN. Menes BL, Han DP et ol. Technical aspects of the Stuphyhmxmr (IL~T~US teichoic acid antibody
assay:
immunoelectrophoretic tion. antigen
selection,
gel diffusion
and counter-
assays,antigen preparaconcentration
effects. and
84
6.
7.
8.
9.
IO.
1 I.
Leading Articles cross-reactions with other organisms, J Clin Microbiol 1981; 13: 2933300. Gemmell CC. King SL, Sturrock RD. Teichoic acid antibody measurement as an aid to the diagnosis of septic arthritis due to Stuphylococcus aureus. Serodiag Immunother Infect Dis 1987; 1: 201-7. Lentino JR, Rytel MW. Detection of circulating free and complexed staphylococcal antigens by enzyme-linked immunosorbent assay. J Clin Microbial 1982; 16: 1019-24. Granstrom M, Julander I, Mollby R. Serological diagnosis of deep Staphylococcus aureus infections by enzyme-linked immunosorbent assay (ELISA) for staphylococcal haemolysins and teichoic acid. Stand J Infect Dis 1983; 41 (Suppl): 132-9. Granstrom M, Julander IG. Hedstrom SA, Mollby R. Enzyme-linked immunosorbent assay for antibodies against teichoic acid in patients with staphylococcal infections. J Clin Microbial 1983; 17: 640-6. Taylor AC, Plommet M. Anti-gamma haemolysin as a diagnostic test in staphylococcal osteomyelitis. J Clin Path 1973; 26: 409-12. Bell JA, Pennington TH. Petrie DT. Western blot
12.
13.
14.
15.
analysis of staphylococcal antibodies present in human sera during health and disease. J Med Microbial 1987; 23: 95-9. Esperson F, Wilkinson BJ, Wheat LJ. White A. Bayer AS, Hopper DC. Antibody response to Slaphylococcus epidermis: comparison of ultrasonic extract and cell wall antigens for serological diagnosis of coagulase-negative staphylococcal endocarditis. Serodiag Immunother Infect Dis 1987; 1: 367 -78. Esperson F. Wheat LJ, Bernis AT, White A, Bayer AS, Hopper DC Solid phase radioimmunoassay for immunoglobulin G antibodies to Staphylococcus epidermidis in serious coagulase-negative staphylococcal infections. Arch Int Med 1987; 147: 689-93. West TE, Cantley JR. Burdash NM. Detection of anti-teichoic acid immunoglobulin G antibodies in experimental Skzphvlococcus epiderntidis endocarditis. Eur J Clin Microbial 1985; 23: 339 42. Ballardie FW, Barsham S, Clutterbuck EJ, Brenchley PE, Bayston R. IgG antibodies to coagulase negative staphylococci in CAPD patients: detection and role as potential opsonins. Adv Perit Dial 1987; 13&7.
Serodiagnosis of staphylococcal infections The usefulness of serological assays in the diagnosis of certain forms of staphylococcal infection has proved to be of some value when a staphylococcal aetiology is suspected but cannot be verified by culture. Measurement of neutralising antibody to the haemolytic activity of staphylococcal a-haemolysin was amongst the earliest methods chosen and for many years was the only method available’. However its applicability was dependent on a stable standardised preparation of the toxin which had been purified to improve its specificity to react with antibody. Being biological in nature, the assay displayed considerable inter-laboratory variation both in its specificity. sensitivity and interpretation. Similar assays which followed, detecting antibody to DNAase. beta-toxin, leucocidin and delta-toxin, suffered from the same criticisms’. Major developments in our knowledge of the structure of the bacterial cell wall, and of the Sraphvlococcus cell wall in particular, have led to the use of two components as antigens for the serological detection of staphylococcal disease’. Peptidoglycan (PG) is the major cell wall component representing about 5&60% of its dry weight. This component forms the basic matrix onto which other substances such as teichoic acid (TA) and protein A are covalently linked. However, only the latter two components are species-specific, whereas PC is present in virtually all procaryotic micro-organisms, although there are differences in fine detail between different species. Teichoic acid in particular has proved useful as an antigen in the detection of antibodies in cases of endocarditis, septicaemia and osteomyelitis caused by Stuphylococcus uureus. Numerous reports exist citing gel diffusion, counter-current immunoelectrophoresis (CIE) radioimmunoassay (RIA) and enzyme-linked immunosorbant assay (ELISA) as methods of choice to detect such antibodies. Raised levels of TA antibodies in endocarditis and septicaemia are a consistent feature of these reports, and it has been suggested that the TA can discriminate between endocarditis and other S. uureus infections, as only sera from S. aureus endocarditis had titres in excess of 4. With a commercially available TA antibody assay4 low levels of antibody were seen in 20% of normal individuals. Tines greater than 4 were considered positive and were regularly
seen in patients with uncomplicated bacteraemia. Despite the many favourable reports 01 TA detection by gel diffusion techniques, certain problems have limited the broader USCof these assays. Various technical aspects of the assays must be carefully controlled to minimise interlaboratory variability and intralaboratory reproducibility’. The sensitivity of the assay in patients with S. crureus endocarditis ranges from 61 to 100%. while the false positive rate in control subjects ranges from zero to 25%. Agar composition and the tcichoic acid content of the antigen used are two critical factors that can influence both the sensitivity and specificity of the assay. One diagnostic problem has been partially solved by the use of methods to detect teichoic acid antibodies. Hitherto. laboratory detection of septic arthritis has been dependent on Gram films, bacteriological culture and leucocyte counts, with varying amounts of success (3550%). However teichoic acid antibodies found by either gel diffusion or counter-current immunoelectrophoresis in either serum or synovial fluid have been successful. Of IO patients with clinical evidence of septic arthritis, four were shown to carry detectable levels of TAA by gel diffusion and another three patients were detected by CIE6. Synovial fluid was not such a suitable specimen in which to look for the antibody. It is likely that any antibody which is present is trapped in immune complexes as a consequence of antigen excess early in the infectious process. In this connection, it has been shown that staphylococcal antigens are present both free and complexed with antibody in various staphylococcal infections (endocarditis, bacteraemia and non-bacteraemic infections such as osteomyelitis). Circulating immune complexes were found in seven of eight patients with staphylococcal bacteraemia’. Thus antigen detection as well as TAA measurements may prove to be the most valuable in distinguishing patients with endocarditis from controls. Another test which may add to the quality of the laboratory detection of anti-staphylococcal antibodies is the parallel measurement of anti alpha-toxin antibodiesa. In an ELISA for antibodies to this toxin and teichoic acid, a 100% positive response was seen in 19 patients with paired serum samples from cases of endocarditis and complicated septicaemia. whilst an 89% rate was detected in uncomplicated bacteraermia. In this way, serological diagnosis of staphylococcal septicaemia and endocarditis
83
Leading Articles could be established in at least 97%“. Antibodies to a-haemolysin are positive in 65% of cases of staphylococcal osteomyelitis. Subsequent application of a test to measure antibody to a-haemolysin increased the detection rate in osteomyelitis to 80%1°. Anti-nuclease has also been tried in this connection giving slightly over 50% positivity in cases of staphylococcal osteomyelitis. Another approach which has been recently evaluated has been immunoblotting (also known as western blotting). By this means the patient’s antibody response to a variety of bacterial antigens can be assessed at one and the same time. Such a bacterial mosaic of antigens is prepared by subjecting whole-cell lysates to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferring them onto nitrocellulose (electroblotting). Any reaction with antibodies present in the patient’s serum can be visualised by an enzyme-linked immunosorbant assay. A study of sera obtained from individuals with osteomyelitis revealed the presence of an antigen of molecular weight 3 1,000 kDa which reacted with IgM antibody. Little or no antibody to this antigen was present in normal human sera”. A similar situation involving coagulasenegative staphylococci as the cause of prosthetic valve endocarditis has not until recently seen any developments in terms of serological measurements of specific antibodies. The clinical significance of a positive blood culture with coagulase-negative staphylococci in patients with prosthetic heart valves and fever can be difficult to determine. A serological assay capable of discriminating between endocarditis and uncomplicated bacteraemia is important since the form of therapy depends upon the in the former long-term antibiotic distinction therapy and/or valve replacement, in the latter short-term antibiotic therapy only. A solid-phase RIA has been developed’? with an ultrasonic cell extract which succeeded in this respect but failed to distinguish between cases of uncomplicated bacteraemia and mere contamination of blood culture bottles with coagulase-negative staphylococci. Later the same group” compared different antigens of S. t~pidertnidis (ultrasonic extract, purified cell walls, peptidoglycan and teichoic acid) for their specificity in diagnosing coagulase-negative staphylococcal endocarditis. However, the purified antigens were no better (or worse) than the ultrasonic extract used previously. This extract
contained at least 43 different antigensId yet was more specific than the purified antigens in an RIA. A quite different approach has been the detection of specific antibodies in patients on chronic ambulatory peritoneal dialysis (CAPD) who develop peritonitis”. An ELlSA with cellwall polysaccharide B as antigen was used such that in seven of I5 peritonitis episodes a rising titre of IgG antibody was measured. No such rise was seen in peritonitis caused by other bacteria. It would then appear that, at least in some instances, CAPD peritonitis due to coagulase-negative staphylococci results in a me;tsurable antibody response. However. even today the serodiagnosis of staphylococcal infections remains a minefield. represen!ed by the variety of antigens that have been used in the detection of specific antigens and the “sappers” who have developed more. and more sophisticated. methods of detection. The frequency with which we are exposed to staphylococci as antigenic stimuli blurs the threshold at which one can distinguish between commensal association and true infection.
References Mudd S. Gladstone GP, Lenhart NA, Hochstcin HD. Titrations of antibodies against alpha-haemolysin and the components of staphylococcal leucocidin in human subjects following immunization. Br J Exp Path 1962; 43: 313-9.
Taylor AG, Fincham WJ. Cook J. Staphylococcal antibodies
in osteomyelitis:
the use ofantisla-
phylococcal nuclease levels in diagnosis. In: Jeljaszewicz J, ed. Staphylococcal infections. Stuttgart:
G. Fischer,
1976: pp 9114.
Oeding P. Natas OB. Fleurette J. Detection of staphylococcal antibodies in human sera. In: MacDonald
A. Smith
G. eds. The staohvlococci.
Aberdeen: Aberdeen University Press: $7 108. Wheat J, Kohler RB, Garten M, White A. Commercially
available
(Endo-Staph)
assay for
tei-
choic acid antibodies. Evaluation in patients with serious Staphylococcus uureuS infections and in controls.
Arch
Intern
Med
1984; 144: 261 4.
Sheagrcn JN. Menes BL, Han DP et ol. Technical aspects of the Stuphyhmxmr (IL~T~US teichoic acid antibody
assay:
immunoelectrophoretic tion. antigen
selection,
gel diffusion
and counter-
assays,antigen preparaconcentration
effects. and
84
6.
7.
8.
9.
IO.
1 I.
Leading Articles cross-reactions with other organisms, J Clin Microbiol 1981; 13: 2933300. Gemmell CC. King SL, Sturrock RD. Teichoic acid antibody measurement as an aid to the diagnosis of septic arthritis due to Stuphylococcus aureus. Serodiag Immunother Infect Dis 1987; 1: 201-7. Lentino JR, Rytel MW. Detection of circulating free and complexed staphylococcal antigens by enzyme-linked immunosorbent assay. J Clin Microbial 1982; 16: 1019-24. Granstrom M, Julander I, Mollby R. Serological diagnosis of deep Staphylococcus aureus infections by enzyme-linked immunosorbent assay (ELISA) for staphylococcal haemolysins and teichoic acid. Stand J Infect Dis 1983; 41 (Suppl): 132-9. Granstrom M, Julander IG. Hedstrom SA, Mollby R. Enzyme-linked immunosorbent assay for antibodies against teichoic acid in patients with staphylococcal infections. J Clin Microbial 1983; 17: 640-6. Taylor AC, Plommet M. Anti-gamma haemolysin as a diagnostic test in staphylococcal osteomyelitis. J Clin Path 1973; 26: 409-12. Bell JA, Pennington TH. Petrie DT. Western blot
12.
13.
14.
15.
analysis of staphylococcal antibodies present in human sera during health and disease. J Med Microbial 1987; 23: 95-9. Esperson F, Wilkinson BJ, Wheat LJ. White A. Bayer AS, Hopper DC. Antibody response to Slaphylococcus epidermis: comparison of ultrasonic extract and cell wall antigens for serological diagnosis of coagulase-negative staphylococcal endocarditis. Serodiag Immunother Infect Dis 1987; 1: 367 -78. Esperson F. Wheat LJ, Bernis AT, White A, Bayer AS, Hopper DC Solid phase radioimmunoassay for immunoglobulin G antibodies to Staphylococcus epidermidis in serious coagulase-negative staphylococcal infections. Arch Int Med 1987; 147: 689-93. West TE, Cantley JR. Burdash NM. Detection of anti-teichoic acid immunoglobulin G antibodies in experimental Skzphvlococcus epiderntidis endocarditis. Eur J Clin Microbial 1985; 23: 339 42. Ballardie FW, Barsham S, Clutterbuck EJ, Brenchley PE, Bayston R. IgG antibodies to coagulase negative staphylococci in CAPD patients: detection and role as potential opsonins. Adv Perit Dial 1987; 13&7.