- Email: [email protected]
TO BLOT OR NOT?
395
the standards of the surgical treatment of this condition. Nevertheless, it is important that cost/benefit studies are done before the technique is generally adopted-a well trained and skilled surgeon, perhaps aided by loupes, may be hard to beat.
TO BLOT OR NOT? IN the
UK, infective endocarditis continues
to
affect
1500 people per annum, of whom some 15-30% will die;1 similar figures have been reported from the United States.2 Culture-negative endocarditis is usually the result of empirical antibiotic use for an undiagnosed febrile episode. It is clinically frustrating and has major implications for patient management despite improved radiological imaging techniques for detecting vegetations; failure to isolate a pathogen necessitates the use of parenteral combination antibiotic therapy with a penicillin and an aminoglycoside for six weeks.3 In patients with penicillin hypersensitivity, vancomycin and an aminoglycoside should be used; this regimen carries an increased risk of toxicity.’ Serological diagnosis of endocarditis based on detection of antibodies in patients’ serum has many attractions. It is the method of choice for Q fever infection, and has been used in the rapid identification of chlamydial, mycoplasma, and certain fungal infections. Serological techniques have also been used to distinguish uncomplicated bloodstream invasion from endocarditis or other deep-seated sepsis caused by Staphylococcus aureus, through detection of antibodies to staphylococcal teichoic acids. Until lately, this serological approach has received little attention for the more usual pathogens responsible for infective endocarditis, such as viridans and faecal streptococci. However, since antibiotic susceptibility and prognosis vary for these infections, development of species-specific serological tests would considerably aid diagnosis and treatment of such
approximately
infections. Various
tests have been studied for the diagnosis of streptococcal endocarditis, including immunofluorescence and enzyme-linked immunoadsorbent assay (ELISA) techniques.6.7 Immunofluorescence is unable to
discriminate between endocarditis or non-endocarditic bloodstream infection or sepsis at an extravascular site, and lends itself only to the measurement of total antibody response, it does
not indicate recent or remote exposure. ELISA techniques have produced some frustrating crossreactions with non-pathogenic organisms.7 Another approach is immunoblotting (also known as western blotting or electroblotting).8 The advantage of this technique over immunofluorescence or ELISA is that it 1.
2
Young SEJ. Aetiology and epidemiology of endocarditis in England and Wales. In: Finch RG, Shanson DC, Littler WA, eds. Infective endocarditis. London: Academic Press, 1987. 7—14. von Reyn CF, Levy BS, Arbeit RD, Fnedland G, Crumpacker CS. Infective endocarditis an analysis based on strict case definitions. Ann Intern Med 1981; 94: 505-18.
to each antigen in a complex mixture of microbial protein antigens to be determined by use of the patient’s own serum as the primary antibody source. The antigen profile obtained provides a fingerprint for the causal organism, thus permitting its identification. Bacterial antigens prepared either as whole-cell lysates or as wall or membrane preparations are initially separated according to molecular weight by sodium dodecyl sulphate
enables the antibody response
polyacrylamide gel electrophoresis (SDS-PAGE). A replica of the polyacrylamide gel is obtained by transferring the proteins electrophoretically from the gel to a nitrocellulose membrane in an immunoblotting tank. After transfer and renaturation of the proteins on to the nitrocellulose, the membrane is treated sequentially with a blocking agent (eg, bovine serum albumin) to block any unbound protein binding sites; the patient’s serum, and a solution containing an enzyme (eg, alkaline phosphatase or horseradish peroxidase) conjugated to antihuman IgG, IgM, or IgA. The blot is then visualised by soaking the membrane in a solution containing a suitable colourless substrate (eg, naphthol AS-MX phosphate/fast red TR salt or 4chloronaphthol/hydrogen peroxide) which, on enzymic activation, deposits a coloured insoluble product on the antigenic site. Immunoblots may also be visualised directly with colloidal-gold-conjugated antihuman immunoglobulins or by autoradiography with 1251-labelled antibodies. Staphylococcal protein A conjugated to alkaline phosphatase, peroxidase, colloidal gold, or 1251 may be used instead of antihuman immunoglobulin conjugates. Although protein A does not distinguish between IgG and IgA and does not detect IgM, its use avoids the problems associated with the cross-reacting antibodies present in the conjugates. The patterns of antibody production as detected by immunoblotting can reliably aid early diagnosis of infective endocarditis, particularly in culture-negative cases. The intensity of the antibody response both to the entire antigen profile and to individual antigens can be quantified by means of scanning densitometry. The information obtained from immunoblotting may indicate recent or remote exposure and permit discrimination between endocarditic and non-endocarditic bloodstream invasion. Evidence for its role in the diagnosis of bacterial endocarditis, including culture-negative infections, has been shown by the groups of Burnie8.9 and Lambert.lO,l1 Immunoblotting is a moderately simple procedure, and the equipment is cheap and certainly within the capabilities of most clinical diagnostic laboratories. By use of commercial mini SDS-PAGE and blotting systems, results can be obtained in 8-10 hours. Although the more accurate immunological definition of the invading organism is a considerable advantage, the technique does not permit antibiotic susceptibility testing, which is of crucial importance. However, by providing a more reliable definition of a pathogen, antibiotic choice based on the predictable susceptibility pattern is possible, thereby perhaps reducing the toxicity and cost of managing culture-negative endocarditis.
3 Pesanti
EL, Smith IM Infective endocarditis with negative blood cultures. An analysis of 52 cases Am J Med 1979; 66: 43-50 4 Scheld WM, Sande MA. Endocarditis and intravascular infections. In Mandell GL, Douglas RG, Bennett JE, eds. Principles and practice ofinfectious diseases. 2nd ed. New York: John Wiley & Sons, 1985. 518. 5 Editorial. Detecting host response to staphylococcal infection. Lancet 1986; i: 953-54. 6 Shanson DC, Kirk N, Humphrey R. Clinical evaluation of a fluorescent antibody test for serological diagnosis of streptococcal endocarditis. J Clin Pathol 1985; 38: 7
92-98 de Rign MG, Bouvet A, Roberts RB. Enzyme-linked immunosorbent assay for the detection of antibodies to nutritionally variant streptococci in patients with endocarditis. J Infect Dis 1986, 153: 116-21
van
JP, Holland M, Matthews RC, Lees W. Role of immunoblotting in the diagnosis of culture negative and enterococcal endocarditis J Clin Pathol 1987; 40:
8 Burnie
1149-58.
J, Matthews RC Immunoblot analysis: a new method for fingerprinting hospital pathogens. J Immunol Meth 1987; 100: 41-46 Aitchison EJ, Lambert PA, Farrell ID. Antigenic composition of an endocarditisassociated isolate of Streptococcus faecalis and identification of its glycoprotein antigens by ligand blotting with lectins. J Med Microbiol 1986; 21: 161-67 Aitchison EJ, Lambert PA, Grace-Smith E, Fararell ID. Serodiagnosis of Streptococcus faecalis by immunoblotting of surface protein antigens. J Clin
9. Burnie
10
11
Microbiol 1987; 25: 211-15
the standards of the surgical treatment of this condition. Nevertheless, it is important that cost/benefit studies are done before the technique is generally adopted-a well trained and skilled surgeon, perhaps aided by loupes, may be hard to beat.
TO BLOT OR NOT? IN the
UK, infective endocarditis continues
to
affect
1500 people per annum, of whom some 15-30% will die;1 similar figures have been reported from the United States.2 Culture-negative endocarditis is usually the result of empirical antibiotic use for an undiagnosed febrile episode. It is clinically frustrating and has major implications for patient management despite improved radiological imaging techniques for detecting vegetations; failure to isolate a pathogen necessitates the use of parenteral combination antibiotic therapy with a penicillin and an aminoglycoside for six weeks.3 In patients with penicillin hypersensitivity, vancomycin and an aminoglycoside should be used; this regimen carries an increased risk of toxicity.’ Serological diagnosis of endocarditis based on detection of antibodies in patients’ serum has many attractions. It is the method of choice for Q fever infection, and has been used in the rapid identification of chlamydial, mycoplasma, and certain fungal infections. Serological techniques have also been used to distinguish uncomplicated bloodstream invasion from endocarditis or other deep-seated sepsis caused by Staphylococcus aureus, through detection of antibodies to staphylococcal teichoic acids. Until lately, this serological approach has received little attention for the more usual pathogens responsible for infective endocarditis, such as viridans and faecal streptococci. However, since antibiotic susceptibility and prognosis vary for these infections, development of species-specific serological tests would considerably aid diagnosis and treatment of such
approximately
infections. Various
tests have been studied for the diagnosis of streptococcal endocarditis, including immunofluorescence and enzyme-linked immunoadsorbent assay (ELISA) techniques.6.7 Immunofluorescence is unable to
discriminate between endocarditis or non-endocarditic bloodstream infection or sepsis at an extravascular site, and lends itself only to the measurement of total antibody response, it does
not indicate recent or remote exposure. ELISA techniques have produced some frustrating crossreactions with non-pathogenic organisms.7 Another approach is immunoblotting (also known as western blotting or electroblotting).8 The advantage of this technique over immunofluorescence or ELISA is that it 1.
2
Young SEJ. Aetiology and epidemiology of endocarditis in England and Wales. In: Finch RG, Shanson DC, Littler WA, eds. Infective endocarditis. London: Academic Press, 1987. 7—14. von Reyn CF, Levy BS, Arbeit RD, Fnedland G, Crumpacker CS. Infective endocarditis an analysis based on strict case definitions. Ann Intern Med 1981; 94: 505-18.
to each antigen in a complex mixture of microbial protein antigens to be determined by use of the patient’s own serum as the primary antibody source. The antigen profile obtained provides a fingerprint for the causal organism, thus permitting its identification. Bacterial antigens prepared either as whole-cell lysates or as wall or membrane preparations are initially separated according to molecular weight by sodium dodecyl sulphate
enables the antibody response
polyacrylamide gel electrophoresis (SDS-PAGE). A replica of the polyacrylamide gel is obtained by transferring the proteins electrophoretically from the gel to a nitrocellulose membrane in an immunoblotting tank. After transfer and renaturation of the proteins on to the nitrocellulose, the membrane is treated sequentially with a blocking agent (eg, bovine serum albumin) to block any unbound protein binding sites; the patient’s serum, and a solution containing an enzyme (eg, alkaline phosphatase or horseradish peroxidase) conjugated to antihuman IgG, IgM, or IgA. The blot is then visualised by soaking the membrane in a solution containing a suitable colourless substrate (eg, naphthol AS-MX phosphate/fast red TR salt or 4chloronaphthol/hydrogen peroxide) which, on enzymic activation, deposits a coloured insoluble product on the antigenic site. Immunoblots may also be visualised directly with colloidal-gold-conjugated antihuman immunoglobulins or by autoradiography with 1251-labelled antibodies. Staphylococcal protein A conjugated to alkaline phosphatase, peroxidase, colloidal gold, or 1251 may be used instead of antihuman immunoglobulin conjugates. Although protein A does not distinguish between IgG and IgA and does not detect IgM, its use avoids the problems associated with the cross-reacting antibodies present in the conjugates. The patterns of antibody production as detected by immunoblotting can reliably aid early diagnosis of infective endocarditis, particularly in culture-negative cases. The intensity of the antibody response both to the entire antigen profile and to individual antigens can be quantified by means of scanning densitometry. The information obtained from immunoblotting may indicate recent or remote exposure and permit discrimination between endocarditic and non-endocarditic bloodstream invasion. Evidence for its role in the diagnosis of bacterial endocarditis, including culture-negative infections, has been shown by the groups of Burnie8.9 and Lambert.lO,l1 Immunoblotting is a moderately simple procedure, and the equipment is cheap and certainly within the capabilities of most clinical diagnostic laboratories. By use of commercial mini SDS-PAGE and blotting systems, results can be obtained in 8-10 hours. Although the more accurate immunological definition of the invading organism is a considerable advantage, the technique does not permit antibiotic susceptibility testing, which is of crucial importance. However, by providing a more reliable definition of a pathogen, antibiotic choice based on the predictable susceptibility pattern is possible, thereby perhaps reducing the toxicity and cost of managing culture-negative endocarditis.
3 Pesanti
EL, Smith IM Infective endocarditis with negative blood cultures. An analysis of 52 cases Am J Med 1979; 66: 43-50 4 Scheld WM, Sande MA. Endocarditis and intravascular infections. In Mandell GL, Douglas RG, Bennett JE, eds. Principles and practice ofinfectious diseases. 2nd ed. New York: John Wiley & Sons, 1985. 518. 5 Editorial. Detecting host response to staphylococcal infection. Lancet 1986; i: 953-54. 6 Shanson DC, Kirk N, Humphrey R. Clinical evaluation of a fluorescent antibody test for serological diagnosis of streptococcal endocarditis. J Clin Pathol 1985; 38: 7
92-98 de Rign MG, Bouvet A, Roberts RB. Enzyme-linked immunosorbent assay for the detection of antibodies to nutritionally variant streptococci in patients with endocarditis. J Infect Dis 1986, 153: 116-21
van
JP, Holland M, Matthews RC, Lees W. Role of immunoblotting in the diagnosis of culture negative and enterococcal endocarditis J Clin Pathol 1987; 40:
8 Burnie
1149-58.
J, Matthews RC Immunoblot analysis: a new method for fingerprinting hospital pathogens. J Immunol Meth 1987; 100: 41-46 Aitchison EJ, Lambert PA, Farrell ID. Antigenic composition of an endocarditisassociated isolate of Streptococcus faecalis and identification of its glycoprotein antigens by ligand blotting with lectins. J Med Microbiol 1986; 21: 161-67 Aitchison EJ, Lambert PA, Grace-Smith E, Fararell ID. Serodiagnosis of Streptococcus faecalis by immunoblotting of surface protein antigens. J Clin
9. Burnie
10
11
Microbiol 1987; 25: 211-15