FEMS MicrobiologyImmunology89 (1992) 105-110 © 1992 Federation of European MicrobiologicalSocieties 0920-8534/92/$05.00 Published by Elsevier
105
FEMSIM 00194
Antibody response to Staphylococcus aureus whole cell, lipase and staphylolysin in patients with S. aureus infections U. R y d i n g 1, j. R e n n e b e r g 2, j. R o l l o f 1 a n d B. C h r i s t e n s s o n 1 1 Department of Infectious Diseases, Lund University Hospital, Sweden, and 2 Department of Bacteriology, General Hospital, MaimS, Sweden
Received 24 April 1991 Revision received 2 September 1991 Accepted 2 October 1991 Key words: S t a p h y l o c o c c u s a u r e u s ; S. a u r e u s whole cell; Lipase; Staphylolysin; ELISA
1. S U M M A R Y Three assays to measure antibodies against a u r e u s whole cells, lipase and staphylolysin were used to try to discriminate between complicated and uncomplicated S. a u reus septicaemia. Sera were examined from 8 patients with S. a u r e u s endocarditis, 23 patients with complicated S. a u r e u s septicaemia, 12 patients with uncomplicated S. a u r e u s septicaemia and 93 febrile non-septicaemic controls. No single assay could distinguish between complicated and uncomplicated S. a u r e u s septicaemia. If the criterion for a positive result is defined as positive antibody level in the anti-lipase ELISA as well as in at least 1 of the other 2 assays, 10/31 patients with S. a u r e u s endocarditis or complicated septicaemia were positive compared to 0 / 9 3 non-septicaemic patients and 0 / 1 2 patients with uncom-
Staphylococcus
Correspondence to: U. Ryding, Department of Infectious Diseases, UniversityHospital S-221 85 Lund, Sweden.
plicated S. a u r e u s septicaemia. Therefore, the combined use of serological assays in the diagnosis of complicated S. a u r e u s septicaemia, one of which is the anti-lipase ELISA, is recommended.
2. I N T R O D U C T I O N When patients with S t a p h y l o c o c c u s a u r e u s septicaemia have an uncomplicated course of infection, antibiotic treatment for 10-14 days is usually sufficient. On the other hand, if the infection is complicated by endocarditis, secondary foci or long-standing primary loci, prolonged antibiotic treatment is generally needed, often in combination with surgical intervention such as draining of infectious foci or heart valve replacement [1]. The prevalence of endocarditis in patients with S. a u r e u s septicaemia varies in different studies from 2 to 24.5% [2-5]. From our experience, about 50% of all S. a u r e u s septicaemia patients have an endocarditis or complicated septicaemia with secondary foci or long-standing primary foci (unpublished results). The clinical diagnosis of compli-
106 cated septicaemia may be difficult to establish. The results from staphylococcal serology has previously been shown to add valuable information [6-9,12-18]. No single serological assay could be used to diagnose all patients with complicated septicaemia, with or without endocarditis, but the combined use of two or more tests was shown to increase both the specificity and the sensitivity of diagnosis [6,7,13,18]. In the present studies three different serological tests with whole S. aureus cells, S. aureus lipase and staphylolysin as antigens were evaluated in the differential diagnosis of complicated and uncomplicated S. aureus septicaemia.
3. M A T E R I A L S AND M E T H O D S 3.1. Patients
337 serum samples from 137 patients were collected at the Department of Infectious Diseases, Lurid University Hospital, Sweden. Acute serum samples were obtained within 10 days after the onset of infection and convalescent sera were obtained between 10-30 days. Convalescent sera were obtained from all patients while acute sera were obtained from all patients in the control group and from 2 9 / 4 4 patients with septicaemia. More than one convalescent serum was obtained from 26 of the patients with septicaemia. Patients with septicaemia had at least two positive blood cultures and an elevated temperature ( > 38.5°C) for at least 2 days. 3.1.1. S. aureus endocarditis. In 7 patients endocarditis was diagnosed by the development of or changes in heart murmur, echocardiography, surgery or autopsy. Another patient with valvular prosthesis and S. aureus septicaemia with longstanding fever was also regarded as endocarditis on clinical grounds. 3.1.2. Complicated S. aureus septicaemia. In 23 patients with complicated septicaemia, there were no signs of endocarditis, but their primary loci were longstanding and not sufficiently drained or they had developed secondary metastatic infections. 3.1.3.
Uncomplicated
S.
aureus septicaemia.
Septicaemia was considered uncomplicated in 12
patients since primary loci, when present, could easily be eradicated within a few days and no signs of secondary metastatic infections developed. 3.1.4. Febrile controls. The control group consisted of 93 patients with fever for reasons other than septicaemia. All these patients had at least two negative blood cultures. 18 patients had pyrexia of unknown origin, 35 patients had pneumonia, 11 had urinary tract infection, 10 had virosis, 6 had wound infection, 5 had gastroenteritis, 4 had erysipelas, 2 had bronchitis, 1 had sarcoidosis and 1 had fever due to infection with Borrelia burgdorferi. 3.2. Antigen production 3.2.1. Whole cell. S. aureus strain NG-143 (a S. aureus Cowan 1 mutant, not presenting protein
A) was grown in Mueller-Hinton broth for 18 h, the viable count reached 108 cfu • m l - 1. This suspension of bacteria was washed 3 times in PBS (pH 7.2), finally diluted to 10 6 c f u ' m 1 - 1 , and used to coat microtitre plates. 3.2.2. Lipase. An extracellular lipase from S. aureus was purified as previously described [10]. 3.2.3. Staphylolysin. Commercially available staphylolysin was provided by the National Bacteriological Laboratory, Stockholm, Sweden. 3.3. Serological assays 3.3.1. Anti-whole cell E L I S A . S. aureus strain
NG-143 ceils 1 x 105 cfu in 100 /~1 PBS were added to each well together with 200 ~1 0.25% (w/v) glutaraldehyde in microtitre plates (NUNC, Immunoplate 1, Denmark). The plates were centrifuged at 21000 X g for 15 min, washed and coated with 200 /zl blocking buffer (PBS (pH 7.2) + Triton X-100 1% ( w / v ) + 1% (w/v) ovalbumin) for 2 h. After a second wash, sera were diluted 1/2000 in blocking buffer and 100 p~l were added to each well for 45 min. The plates were washed extensively with PBS. Peroxidase conjugated antihuman IgG (Dakopatts, Denmark) diluted 1/2000 in 100 ~1 blocking buffer was added to each well for 45 rain. After extensive washing, 100 /~1 of substrate (1-2-phenylenediamine-dihydrochloride (Fluka, Sweden) + hydrogen peroxide) were added to each well. The
107
reaction was stopped with 1 M H 2 S O 4 after 25 rain, and the plates were read at 450 nm in a spectrophotometer (Micro E L I S A reader, Dynatech Laboratories Inc., ME, U.S.A.). Sera were tested in triplicate and mean values were calculated. Each plate contained two control sera with high and low antibody levels, respectively, which were used to calculate an E L I S A index: A 450 nm value high control A 450 nm value low control To avoid significant day to day inter-assay variation, only plates giving a ratio of 2.0-2.5 between the two controls were included. For each serum an E L i S A index was calculated: A 450 nm value test serum A 450 nm value low control 3.3.2. Anti-lipase ELISA. A previously described anti-lipase E L I S A [8] was slightly modified. Antibody levels were recorded as ELISA-index units where the mean A 405 nm value of a serum sample run in duplicate, was divided by the mean A 405 nm values of a standard serum run in quadruplicate on each microtitre plate. The absorbance value obtained in wells lacking antigen but otherwise identical were subtracted from all other values. 3.3.3. Determination of antibodies to staphylococcal alpha-toxin (ASTA). The A S T A test in a tube-dilution method is a well-established assay. It was first described by Neisser and Wechsberg in 1901 [11]. The assay was done as described by them but modified to a micromethod. The patients serum (50/~1) was added to each well in a microtitre plate. Alpha-toxin (50 p~l) in standard dilutions from 0.9 to 10 A S T A U . m l -~ was added. Finally 25 /zl of 2% ( v / v ) rabbit erythrocytes suspended in 0.15 M NaC1 were added. After incubation over-night plates were read for inhibition of haemolysis.
control group. This gave an upper normal E L I S A index of 2.762 in the whole cell ELISA, 0.587 in the anti-lipase E L I S A and 3.26 in the ASTA. The results were regarded as positive when the test p e a k value, obtained more than 10 days after onset of infection, was greater than the upper normal antibody level. Due to lack of acute sera in 15 patients with S. aureus septicaemia, titre changes were not calculated. When the antibody level in more than one convalescent serum was measured, only the peak value was recorded.
4.1. Antibodies to whole cells Peak I g G antibody levels in patients with S. aureus endocarditis, complicated septicaemia, uncomplicated septicaemia and febrile controls are shown in Fig. l. In 1 / 8 patients (12%) with endocarditis, 6 / 2 3 patients (26%) with complicated septicaemia and 4 / 1 2 patients (33%) with uncomplicated septicaemia, positive antibody levels were found. None of the 93 febrile controls were positive.
f
105
FEMSIM 00194
Antibody response to Staphylococcus aureus whole cell, lipase and staphylolysin in patients with S. aureus infections U. R y d i n g 1, j. R e n n e b e r g 2, j. R o l l o f 1 a n d B. C h r i s t e n s s o n 1 1 Department of Infectious Diseases, Lund University Hospital, Sweden, and 2 Department of Bacteriology, General Hospital, MaimS, Sweden
Received 24 April 1991 Revision received 2 September 1991 Accepted 2 October 1991 Key words: S t a p h y l o c o c c u s a u r e u s ; S. a u r e u s whole cell; Lipase; Staphylolysin; ELISA
1. S U M M A R Y Three assays to measure antibodies against a u r e u s whole cells, lipase and staphylolysin were used to try to discriminate between complicated and uncomplicated S. a u reus septicaemia. Sera were examined from 8 patients with S. a u r e u s endocarditis, 23 patients with complicated S. a u r e u s septicaemia, 12 patients with uncomplicated S. a u r e u s septicaemia and 93 febrile non-septicaemic controls. No single assay could distinguish between complicated and uncomplicated S. a u r e u s septicaemia. If the criterion for a positive result is defined as positive antibody level in the anti-lipase ELISA as well as in at least 1 of the other 2 assays, 10/31 patients with S. a u r e u s endocarditis or complicated septicaemia were positive compared to 0 / 9 3 non-septicaemic patients and 0 / 1 2 patients with uncom-
Staphylococcus
Correspondence to: U. Ryding, Department of Infectious Diseases, UniversityHospital S-221 85 Lund, Sweden.
plicated S. a u r e u s septicaemia. Therefore, the combined use of serological assays in the diagnosis of complicated S. a u r e u s septicaemia, one of which is the anti-lipase ELISA, is recommended.
2. I N T R O D U C T I O N When patients with S t a p h y l o c o c c u s a u r e u s septicaemia have an uncomplicated course of infection, antibiotic treatment for 10-14 days is usually sufficient. On the other hand, if the infection is complicated by endocarditis, secondary foci or long-standing primary loci, prolonged antibiotic treatment is generally needed, often in combination with surgical intervention such as draining of infectious foci or heart valve replacement [1]. The prevalence of endocarditis in patients with S. a u r e u s septicaemia varies in different studies from 2 to 24.5% [2-5]. From our experience, about 50% of all S. a u r e u s septicaemia patients have an endocarditis or complicated septicaemia with secondary foci or long-standing primary foci (unpublished results). The clinical diagnosis of compli-
106 cated septicaemia may be difficult to establish. The results from staphylococcal serology has previously been shown to add valuable information [6-9,12-18]. No single serological assay could be used to diagnose all patients with complicated septicaemia, with or without endocarditis, but the combined use of two or more tests was shown to increase both the specificity and the sensitivity of diagnosis [6,7,13,18]. In the present studies three different serological tests with whole S. aureus cells, S. aureus lipase and staphylolysin as antigens were evaluated in the differential diagnosis of complicated and uncomplicated S. aureus septicaemia.
3. M A T E R I A L S AND M E T H O D S 3.1. Patients
337 serum samples from 137 patients were collected at the Department of Infectious Diseases, Lurid University Hospital, Sweden. Acute serum samples were obtained within 10 days after the onset of infection and convalescent sera were obtained between 10-30 days. Convalescent sera were obtained from all patients while acute sera were obtained from all patients in the control group and from 2 9 / 4 4 patients with septicaemia. More than one convalescent serum was obtained from 26 of the patients with septicaemia. Patients with septicaemia had at least two positive blood cultures and an elevated temperature ( > 38.5°C) for at least 2 days. 3.1.1. S. aureus endocarditis. In 7 patients endocarditis was diagnosed by the development of or changes in heart murmur, echocardiography, surgery or autopsy. Another patient with valvular prosthesis and S. aureus septicaemia with longstanding fever was also regarded as endocarditis on clinical grounds. 3.1.2. Complicated S. aureus septicaemia. In 23 patients with complicated septicaemia, there were no signs of endocarditis, but their primary loci were longstanding and not sufficiently drained or they had developed secondary metastatic infections. 3.1.3.
Uncomplicated
S.
aureus septicaemia.
Septicaemia was considered uncomplicated in 12
patients since primary loci, when present, could easily be eradicated within a few days and no signs of secondary metastatic infections developed. 3.1.4. Febrile controls. The control group consisted of 93 patients with fever for reasons other than septicaemia. All these patients had at least two negative blood cultures. 18 patients had pyrexia of unknown origin, 35 patients had pneumonia, 11 had urinary tract infection, 10 had virosis, 6 had wound infection, 5 had gastroenteritis, 4 had erysipelas, 2 had bronchitis, 1 had sarcoidosis and 1 had fever due to infection with Borrelia burgdorferi. 3.2. Antigen production 3.2.1. Whole cell. S. aureus strain NG-143 (a S. aureus Cowan 1 mutant, not presenting protein
A) was grown in Mueller-Hinton broth for 18 h, the viable count reached 108 cfu • m l - 1. This suspension of bacteria was washed 3 times in PBS (pH 7.2), finally diluted to 10 6 c f u ' m 1 - 1 , and used to coat microtitre plates. 3.2.2. Lipase. An extracellular lipase from S. aureus was purified as previously described [10]. 3.2.3. Staphylolysin. Commercially available staphylolysin was provided by the National Bacteriological Laboratory, Stockholm, Sweden. 3.3. Serological assays 3.3.1. Anti-whole cell E L I S A . S. aureus strain
NG-143 ceils 1 x 105 cfu in 100 /~1 PBS were added to each well together with 200 ~1 0.25% (w/v) glutaraldehyde in microtitre plates (NUNC, Immunoplate 1, Denmark). The plates were centrifuged at 21000 X g for 15 min, washed and coated with 200 /zl blocking buffer (PBS (pH 7.2) + Triton X-100 1% ( w / v ) + 1% (w/v) ovalbumin) for 2 h. After a second wash, sera were diluted 1/2000 in blocking buffer and 100 p~l were added to each well for 45 min. The plates were washed extensively with PBS. Peroxidase conjugated antihuman IgG (Dakopatts, Denmark) diluted 1/2000 in 100 ~1 blocking buffer was added to each well for 45 rain. After extensive washing, 100 /~1 of substrate (1-2-phenylenediamine-dihydrochloride (Fluka, Sweden) + hydrogen peroxide) were added to each well. The
107
reaction was stopped with 1 M H 2 S O 4 after 25 rain, and the plates were read at 450 nm in a spectrophotometer (Micro E L I S A reader, Dynatech Laboratories Inc., ME, U.S.A.). Sera were tested in triplicate and mean values were calculated. Each plate contained two control sera with high and low antibody levels, respectively, which were used to calculate an E L I S A index: A 450 nm value high control A 450 nm value low control To avoid significant day to day inter-assay variation, only plates giving a ratio of 2.0-2.5 between the two controls were included. For each serum an E L i S A index was calculated: A 450 nm value test serum A 450 nm value low control 3.3.2. Anti-lipase ELISA. A previously described anti-lipase E L I S A [8] was slightly modified. Antibody levels were recorded as ELISA-index units where the mean A 405 nm value of a serum sample run in duplicate, was divided by the mean A 405 nm values of a standard serum run in quadruplicate on each microtitre plate. The absorbance value obtained in wells lacking antigen but otherwise identical were subtracted from all other values. 3.3.3. Determination of antibodies to staphylococcal alpha-toxin (ASTA). The A S T A test in a tube-dilution method is a well-established assay. It was first described by Neisser and Wechsberg in 1901 [11]. The assay was done as described by them but modified to a micromethod. The patients serum (50/~1) was added to each well in a microtitre plate. Alpha-toxin (50 p~l) in standard dilutions from 0.9 to 10 A S T A U . m l -~ was added. Finally 25 /zl of 2% ( v / v ) rabbit erythrocytes suspended in 0.15 M NaC1 were added. After incubation over-night plates were read for inhibition of haemolysis.
control group. This gave an upper normal E L I S A index of 2.762 in the whole cell ELISA, 0.587 in the anti-lipase E L I S A and 3.26 in the ASTA. The results were regarded as positive when the test p e a k value, obtained more than 10 days after onset of infection, was greater than the upper normal antibody level. Due to lack of acute sera in 15 patients with S. aureus septicaemia, titre changes were not calculated. When the antibody level in more than one convalescent serum was measured, only the peak value was recorded.
4.1. Antibodies to whole cells Peak I g G antibody levels in patients with S. aureus endocarditis, complicated septicaemia, uncomplicated septicaemia and febrile controls are shown in Fig. l. In 1 / 8 patients (12%) with endocarditis, 6 / 2 3 patients (26%) with complicated septicaemia and 4 / 1 2 patients (33%) with uncomplicated septicaemia, positive antibody levels were found. None of the 93 febrile controls were positive.
f
