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Pneumococcal antigens in sputa: ELISA for the detection of pneumococcal C-polysaccharide in sputa from pneumonia patients
DIAGNMICROBIOLINFECTDIS 1987;7:73-75
73
Pneumococcal Antigens in Sputa: ELISA for the Detection of Pneumococcal C-Polysaccharide in Sputa from Pneumonia Patients Aud Krook and Hans Holmberg
An improved ELISA, the LKB UltroBact Pneumococcus Kit detecting pneumococcal C-polysaccharide, has been tested. Sputum samples from 72 patients with community acquired pneumonia were included in the study. The sensitivity obtained was 96.1% and the specificity 92.6%. This ELISA might offer a useful diagnostic method in major clinical microbiolagic laboratories for demonstrating Streptococcus pneumonia in sputa from patients with pneumonia.
Pneumococci are the most common cause of pneumonia of hospitalized patients (Donowitz and Mandell, 1985). A rapid diagnosis is important for the right choice of antibiotic treatment. In adult patients a presumptive diagnosis can be obtained by demonstrating Streptococcus p n e u m o n i a e in expectorated sputum samples (Bartlett and Finegold, 1978). We have earlier described an enzyme-linked immunosorbent assay (ELISA) for this purpose (Holmberg et al., 1985). However, the disadvantage with the previous test was the time required, 4-5 hr, which seems too long for routine diagnostic work. Therefore, a new kit has been developed, which is read after approximately 2 hr, and we have tested it on sputa from pneumonia patients. A 6-mo study of adult patients with community acquired pneumonia was performed from November 1984 to April 1985 at the Department of Infectious Diseases, (3rebro Medical Center Hospital, (~rebro, Sweden. A total of 86 patients with clinical and radiologic evidence for pneumonia was accepted for the study. Samples from blood, sputum, and nasopharynx were taken at the admission to the ward. Presumptive etiology of the patients' pneumonias is presented in Table 1. Sputum culture was performed in a semiquantitative manner. The samples were homogenized with an equal volume of Sputolysin (Calbiochem, Lucerne, Switzerland), shaken on a Vortex mixer for 5 min, and then incubated at room temperature for another 15 min. Of these partially or totally homogenized sputa, 10-p.1 portions were taken with a loop, cultured on hematin agar with a 50-~,g oleandomycin disk, and incubated in moist air with 5% CO2. In this procedure one colony represents 2 × 102 CPU/ml. Another sample was diluted 1 : 100 with nutrient broth (Oxoid Ltd., Basingstoke, UK). Portions (1 p,1) of this dilution were cultured on hematin agar as described
From the Department of Infectious Diseases, Roslagstulls Hospital, Karolinska Institute, Stockholm and the Department of Infectious Diseases, Orebro Medical Center Hospital, Orebro, Sweden. Address requests for reprints to: Aud Krook, Department of Infectious Diseases, Roslagstulls Hospital, Karolinska Institute, Box 5651, S-114 89 Stockholm, Sweden. Received September 9, 1986; revised and accepted December 19, 1986. © 1987Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue,New York, NY 10017
0732-8893/87/$3.50
74
A. Krook and H. Holmberg
TABLE 1. Presumptive Etiology of C o m m u n i t y Acquired P n e u m o n i a in 86 Patients: The Basis for Etiologic Diagnosis is Indicated for each Patient Organism
n
Streptococcus p n e u m o n i a e Sa 1 7 b 3c Streptococcus pneumoniae b + Hemophilus influenzae b Streptococcus pneumoniae 2a 2b + influenza virus Bd Mycoplosma pneumoniae d Mycoplasma pneumoniae a + Streptococcus group Cb Chlamydio psittaci d Haemophilus influenzae 4b 1¢ Haemophilus influenzae b + virusa Pseudomonas aeruginosa b
25
1
4 16 1 5 5
2 1
Streptococcus gr Gb Influenza virus Ad Influenza virus Bd
1 3 2
Known etiology (77%) Unknown etiology (23%)
66 20
°Isolated in blood. blsolated in sputum and not in blood. Clsolated in nasopharynxonly. dPositive serology. above, on bovine blood agar with gentian violet provided with two paper disks containing 0.2 IU of bacitracin and 6 ~g of optochin, respectively, incubated in moist air with 5% CO2, and anaerobically (GasPak; BBL Microbiology Systems, Cockeysville, Md) on b r a i n - h e a r t infusion-blood agar (5% bovine blood) s u p p l e m e n t e d with yeast extract, v i t a m i n K, and h e m i n (4 ~g/ml) provided with an optochin paper disk. With this d i l u t i o n one colony represents 2 x 105 CFU/ml. Other details for the diagnostic procedures were also the same as i n the earlier study (Holmberg et el., 1985). TABLE 2. Pneumococcal Antigen Detection in S p u t u m by ELISA i n 72 P n e u m o n i a Patients: The r e m a i n i n g 14 patients were not able to deliver a s p u t u m Group
Description
la lb
S. pneumonioe isolated in sputumb S. pneumoniae isolated in blood and not in sputumc S. pneumonioe isolated in nasopharynx onlyd No S. pneumoniae isolated in sputum, blood or nasopharynx; patients with pneumonia of unknown etiology No S. pneumoniae isolated in sputum, blood or nasopharynx; patients with pneumonia of other known or suspected etiology
Sputum samples (n}a -[-
lc 2
3
25 (0) 0
m
1 (0) 1 (0)
2 (0)
0
7 (0)
9 (0)
2 (0}
25 (8)
°Numberof patients given antibiotic treatmentbefore admissionto the ward are given in parentheses. bTypes (numberof isolates when greater than one is given in parentheses):3(7), 4, 6A(3), 6B(3}, 7F(3), 10A, 11A, 14(2},17, 19F(2], 31, 35F. q'ype 14. dTypes 7F, 9N.
Notes
75
The ELISA used in the present study was the LKB UltroBact Pneumococcus Kit (LKB-Produkter AB, Box 305, S-161 26 Bromma, Sweden), a modified sandwich ELISA with a monoclonal antibody directed against phosphorylcholine, a major antigen of the pneumococcal C-polysaccharide (PnC), and an affinity purified polyclonal rabbit anti-PnC antibody in the test system. The results are presented in Table 2. In the patients with pneumococci isolated in sputum (group la) 25 of 26 were positive in the test (sensitivity 96.1%). No patients in this group were pretreated with antibiotics. In the patients with pneumonia of other known or suspected etiology (group 3) 25 of 27 patients were negative (specificity 92.6%) of which eight were pretreated with antibiotics and the treatment not successful. In the patients with pneumonia of unknown etiology seven of 16 patients (43.8%) were positive and no one was pretreated. Fourteen patients were not able to deliver a sputum sample. An etiologic diagnosis could be achieved in most of the patients with pneumococcal pneumonia. The two false positive samples were from patients in which Haemophilus influenzae and Pseudomonas aeruginosa, respectively, had been isolated from the sputa. Although crossreactions cannot be excluded, a double infection with pneumococci is more probable as both patients recovered quickly on penicillin treatment alone. Of the patients with pneumonia of unknown etiology, as many as 43.8% were positive in the test. Four of the seven patients who were positive in the test were treated with penicillin and their fever had decreased below 38°C in 1 day, which gave a strong suggestion for pneumococcal etiology. The other three patients were treated with tetracycline and ampicilline respectively and had a fever decrease below 38°C in 1-5 days. The LKB UltroBact Pneumococcus Kit, in our opinion, is a sensitive and specific test for demonstrating S. pneumoniae in sputa from patients with pneumonia and might offer a useful diagnostic method, especially in greater clinical microbiologic laboratories. Supported by the Swedish Board for Technical Development and LKB-Produkter AB, Bromma, Sweden. We thank Elisabeth Mansfeld (Department of Clinical Microbiologyand Immunology, Orebro Medical Center Hospital) for her skilfull laboratory assistance.
REFERENCES Donowitz GR, Mandell GL (1985) Acute pneumonia. In Principles and Practice of Infectious Diseases. GL Mandell, RG Douglas, JE Bennet Eds., New York: Wiley Medical, p. 400. Bartlett JG, Finegold SM (1978) Bacteriology of expectorated sputum with quantitative culture and wash technique compared to transtracheal aspirates. Am Rev Resp Dis 117:1019-1027. Holmberg H, Holme T, Krook A, Olsson T, Sj6berg L, Sj6gren AM (1985) Detection of C polysaccharide in Streptococcus pneumoniae in sputa of pneumonia patients by an enzymelinked immunosorbent assay. J Clin Microbiol 22:111-115.
73
Pneumococcal Antigens in Sputa: ELISA for the Detection of Pneumococcal C-Polysaccharide in Sputa from Pneumonia Patients Aud Krook and Hans Holmberg
An improved ELISA, the LKB UltroBact Pneumococcus Kit detecting pneumococcal C-polysaccharide, has been tested. Sputum samples from 72 patients with community acquired pneumonia were included in the study. The sensitivity obtained was 96.1% and the specificity 92.6%. This ELISA might offer a useful diagnostic method in major clinical microbiolagic laboratories for demonstrating Streptococcus pneumonia in sputa from patients with pneumonia.
Pneumococci are the most common cause of pneumonia of hospitalized patients (Donowitz and Mandell, 1985). A rapid diagnosis is important for the right choice of antibiotic treatment. In adult patients a presumptive diagnosis can be obtained by demonstrating Streptococcus p n e u m o n i a e in expectorated sputum samples (Bartlett and Finegold, 1978). We have earlier described an enzyme-linked immunosorbent assay (ELISA) for this purpose (Holmberg et al., 1985). However, the disadvantage with the previous test was the time required, 4-5 hr, which seems too long for routine diagnostic work. Therefore, a new kit has been developed, which is read after approximately 2 hr, and we have tested it on sputa from pneumonia patients. A 6-mo study of adult patients with community acquired pneumonia was performed from November 1984 to April 1985 at the Department of Infectious Diseases, (3rebro Medical Center Hospital, (~rebro, Sweden. A total of 86 patients with clinical and radiologic evidence for pneumonia was accepted for the study. Samples from blood, sputum, and nasopharynx were taken at the admission to the ward. Presumptive etiology of the patients' pneumonias is presented in Table 1. Sputum culture was performed in a semiquantitative manner. The samples were homogenized with an equal volume of Sputolysin (Calbiochem, Lucerne, Switzerland), shaken on a Vortex mixer for 5 min, and then incubated at room temperature for another 15 min. Of these partially or totally homogenized sputa, 10-p.1 portions were taken with a loop, cultured on hematin agar with a 50-~,g oleandomycin disk, and incubated in moist air with 5% CO2. In this procedure one colony represents 2 × 102 CPU/ml. Another sample was diluted 1 : 100 with nutrient broth (Oxoid Ltd., Basingstoke, UK). Portions (1 p,1) of this dilution were cultured on hematin agar as described
From the Department of Infectious Diseases, Roslagstulls Hospital, Karolinska Institute, Stockholm and the Department of Infectious Diseases, Orebro Medical Center Hospital, Orebro, Sweden. Address requests for reprints to: Aud Krook, Department of Infectious Diseases, Roslagstulls Hospital, Karolinska Institute, Box 5651, S-114 89 Stockholm, Sweden. Received September 9, 1986; revised and accepted December 19, 1986. © 1987Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue,New York, NY 10017
0732-8893/87/$3.50
74
A. Krook and H. Holmberg
TABLE 1. Presumptive Etiology of C o m m u n i t y Acquired P n e u m o n i a in 86 Patients: The Basis for Etiologic Diagnosis is Indicated for each Patient Organism
n
Streptococcus p n e u m o n i a e Sa 1 7 b 3c Streptococcus pneumoniae b + Hemophilus influenzae b Streptococcus pneumoniae 2a 2b + influenza virus Bd Mycoplosma pneumoniae d Mycoplasma pneumoniae a + Streptococcus group Cb Chlamydio psittaci d Haemophilus influenzae 4b 1¢ Haemophilus influenzae b + virusa Pseudomonas aeruginosa b
25
1
4 16 1 5 5
2 1
Streptococcus gr Gb Influenza virus Ad Influenza virus Bd
1 3 2
Known etiology (77%) Unknown etiology (23%)
66 20
°Isolated in blood. blsolated in sputum and not in blood. Clsolated in nasopharynxonly. dPositive serology. above, on bovine blood agar with gentian violet provided with two paper disks containing 0.2 IU of bacitracin and 6 ~g of optochin, respectively, incubated in moist air with 5% CO2, and anaerobically (GasPak; BBL Microbiology Systems, Cockeysville, Md) on b r a i n - h e a r t infusion-blood agar (5% bovine blood) s u p p l e m e n t e d with yeast extract, v i t a m i n K, and h e m i n (4 ~g/ml) provided with an optochin paper disk. With this d i l u t i o n one colony represents 2 x 105 CFU/ml. Other details for the diagnostic procedures were also the same as i n the earlier study (Holmberg et el., 1985). TABLE 2. Pneumococcal Antigen Detection in S p u t u m by ELISA i n 72 P n e u m o n i a Patients: The r e m a i n i n g 14 patients were not able to deliver a s p u t u m Group
Description
la lb
S. pneumonioe isolated in sputumb S. pneumoniae isolated in blood and not in sputumc S. pneumonioe isolated in nasopharynx onlyd No S. pneumoniae isolated in sputum, blood or nasopharynx; patients with pneumonia of unknown etiology No S. pneumoniae isolated in sputum, blood or nasopharynx; patients with pneumonia of other known or suspected etiology
Sputum samples (n}a -[-
lc 2
3
25 (0) 0
m
1 (0) 1 (0)
2 (0)
0
7 (0)
9 (0)
2 (0}
25 (8)
°Numberof patients given antibiotic treatmentbefore admissionto the ward are given in parentheses. bTypes (numberof isolates when greater than one is given in parentheses):3(7), 4, 6A(3), 6B(3}, 7F(3), 10A, 11A, 14(2},17, 19F(2], 31, 35F. q'ype 14. dTypes 7F, 9N.
Notes
75
The ELISA used in the present study was the LKB UltroBact Pneumococcus Kit (LKB-Produkter AB, Box 305, S-161 26 Bromma, Sweden), a modified sandwich ELISA with a monoclonal antibody directed against phosphorylcholine, a major antigen of the pneumococcal C-polysaccharide (PnC), and an affinity purified polyclonal rabbit anti-PnC antibody in the test system. The results are presented in Table 2. In the patients with pneumococci isolated in sputum (group la) 25 of 26 were positive in the test (sensitivity 96.1%). No patients in this group were pretreated with antibiotics. In the patients with pneumonia of other known or suspected etiology (group 3) 25 of 27 patients were negative (specificity 92.6%) of which eight were pretreated with antibiotics and the treatment not successful. In the patients with pneumonia of unknown etiology seven of 16 patients (43.8%) were positive and no one was pretreated. Fourteen patients were not able to deliver a sputum sample. An etiologic diagnosis could be achieved in most of the patients with pneumococcal pneumonia. The two false positive samples were from patients in which Haemophilus influenzae and Pseudomonas aeruginosa, respectively, had been isolated from the sputa. Although crossreactions cannot be excluded, a double infection with pneumococci is more probable as both patients recovered quickly on penicillin treatment alone. Of the patients with pneumonia of unknown etiology, as many as 43.8% were positive in the test. Four of the seven patients who were positive in the test were treated with penicillin and their fever had decreased below 38°C in 1 day, which gave a strong suggestion for pneumococcal etiology. The other three patients were treated with tetracycline and ampicilline respectively and had a fever decrease below 38°C in 1-5 days. The LKB UltroBact Pneumococcus Kit, in our opinion, is a sensitive and specific test for demonstrating S. pneumoniae in sputa from patients with pneumonia and might offer a useful diagnostic method, especially in greater clinical microbiologic laboratories. Supported by the Swedish Board for Technical Development and LKB-Produkter AB, Bromma, Sweden. We thank Elisabeth Mansfeld (Department of Clinical Microbiologyand Immunology, Orebro Medical Center Hospital) for her skilfull laboratory assistance.
REFERENCES Donowitz GR, Mandell GL (1985) Acute pneumonia. In Principles and Practice of Infectious Diseases. GL Mandell, RG Douglas, JE Bennet Eds., New York: Wiley Medical, p. 400. Bartlett JG, Finegold SM (1978) Bacteriology of expectorated sputum with quantitative culture and wash technique compared to transtracheal aspirates. Am Rev Resp Dis 117:1019-1027. Holmberg H, Holme T, Krook A, Olsson T, Sj6berg L, Sj6gren AM (1985) Detection of C polysaccharide in Streptococcus pneumoniae in sputa of pneumonia patients by an enzymelinked immunosorbent assay. J Clin Microbiol 22:111-115.