Vol. 30, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1992, p. 517-519
0095-1137/92/020517-03$02.00/0 Copyright C 1992, American Society for Microbiology
Minimum Number of Pneumococci Required for Capsular Antigen To Be Detectable by Latex Agglutination YVETTE HOLLOWAY,'* WILLEM G. BOERSMA,2 HENRIETTE KUTTSCHRUTTER,l AND JAN A. M. SNIJDER1 Immunology Research, Regional Public Health Laboratory,' and Department of Pulmonary Diseases, University Hospital,2 Groningen, The Netherlands Received 29 July 1991/Accepted 5 November 1991
Forty-eight strains of Streptococcus pneumoniae were tested in vitro to determine the minimum number required for pneumococcal capsular antigen to be detectable by latex agglutination. It was found that 106 to 107 microorganisms per ml were needed and that antigen remained detectable even when viable pneumococci could no longer be demonstrated.
Pneumococcal capsular antigen has been detected by many methods, and all of these have a threshold of detection, with the lowest reported range running from 103/ml for enzyme-linked immunosorbent assay (26) and counterimmunoelectrophoresis (CIE; 13) and an average of 106/ml for these and other methods (2-4, 6, 9, 10, 15, 18, 22). In most instances, Omniserum (Staten Serum Institut, Copenhagen, Denmark) was used, but some workers, who obtained lower thresholds, used species-specific cell wall antiserum (26) or type-specific antiserum (9). With the latter, chances of detecting a single type of pneumococcus are better than they are with Omniserum (14). In order to establish the threshold number of pneumococci required for capsular antigen to be detectable by latex agglutination (LA), 48 pneumococci isolated from 35 patients were tested. The sources and types are listed in Table 1. Pneumococci were removed from blood agar plates with a cotton swab, and serial dilutions of 10-1 to 10' were prepared in physiological saline. These suspensions were divided into two parts: one to be used for antigen detection and one for quantitative cultures. Pneumococcal capsular antigen detection was performed by LA, using the Wellcogen Streptococcus pneumoniae Kit (Wellcome Diagnostics, Dartford, England) according to the manufacturer's instructions. The second part of the dilution row was used for estimation of the number of pneumococci present in each tube. Tubes were vortexed, and 25 ,ul from each tube was spread out evenly on a blood agar plate by using bent glass rods. The number of pneumococci per milliliter in each tube was calculated by multiplying the number of colonies grown by the dilution factor of that tube and then by the additional dilution factor of an inoculum of 25 RIu. For growth curves, three serial dilutions (10-3, 10', and 10-7 pneumococci per ml) were prepared in brain heart infusion (BHI) broth. Suspensions were vortexed, 500 p,l was removed from each tube for antigen detection, and 25 ,ud was removed for quantitative culture as described above. This process was repeated at 4, 8, 24, 48, and 72 h. BHI tubes were incubated at 37°C in 5% CO2 until growth curves were completed. It was found that approximately 106 to 107 pneumococci per ml were required for pneumococcal capsular antigen to
be detectable by LA directly from blood plates. Tubes containing fewer pneumococci per milliliter did not yield agglutination by LA. This number of pneumococci was required regardless of the source, the patient category, or the capsular type. Results for capsular types are listed in Table 2. In growth curves, pneumococcal capsular antigen could not be detected until the microorganisms had reached a concentration of approximately 106 to 107/ml in BHI broth. Typical examples are shown in Fig. 1. Once the concentration of 106 to 107 pneumococci per ml was reached, antigen remained detectable for the entire 72 h of the experiment, even when the number of pneumococci fell below this number or when no further viable pneumococci could be detected (Fig. 2). On the whole, CIE tends to react earlier than does LA, but this is probably because LA detects an antigen which is different from that detected in CIE (25). It is reported elsewhere that detectable levels of antigen are related to the logarithmic phase of growth (9), but we were unable to confirm this. Pneumococci well into the logarithmic phase (Fig. 1) still did not produce detectable antigen until the required number was attained, and microorganisms that were dying or dead (Fig. 2) still produced detectable antigen providing the threshold had previously been reached. An in vitro experiment such as ours cannot be entirely correlated to the in vivo situation because pneumococcal
10 m I C R
0 0 R G A N I S M S
10
10 10
Type 23 Type 7
a
Type 3
7
10 6
10 10
/
10
L
10
6
W-,o
4 3
0 2 4 6 *
Type 19
9
0 2 4 6 8 o0 2 4 TIME (HOURS)
0 2 4 6 8
FIG. 1. Pneumococcal capsular antigen detection in BHI broth. Symbols: 0, no antigen detectable; *, antigen detectable.
Corresponding author. 517
518
NOTES
J. CLIN. MICROBIOL. TABLE 1. Types and origins of 48 pneumococci from 35 patients No. of isolates
Source (n)
Category of patients (n)
3
4
5 6 7 9 10 11 13 14
1 1 2 1 1 2 6 3
18 19
1 9
Blood (1), throat (1), sputum (1) Blood (1) Sputum (1) Sputum (1) Blood (1), sputum (1) Blood (1) Sputum (1) Throat (1), sputum (1) Sputum (6) Blood (2) Sputum (1) CSFd (1) Bronchial washings (2), throat (1), sputum (3) Blood (1) Sputum (1) Sputum (1) Sputum (2) Throat (1) Sputum (1) Sputum (1) Nose (2), throat (8), saliva (2)
Pneumococcal pneumonia (2a) Pneumococcal meningitis (1) COPD with LRTIb (1) COPD with LRTI (1) Pneumococcal pneumonia (2) Pneumococcal pneumonia (1) COPD with LRTI (1) Pneumococcal pneumonia (1a) Pneumococcal pneumonia (1C) Pneumococcal pneumonia (2) COPD with LRTI (1) Pneumococcal meningitis (1) COPD with LRTI (3Y) Pneumococcal pneumonia (1) Pneumococcal pneumonia (1) LRTI (1) Pneumococcal pneumonia (1a) Pneumococcal pneumonia (1) Pneumococcal pneumonia (1) COPD with LRTI (1) COPD out-patients (10f)
Pneumococcal type
22 23 31 Untyped
2 1 1 13
a Two pneumococci from the same patient on different days. b COPD, chronic obstructive pulmonary disease; LRTI, lower respiratory tract infection. c Six sputum samples from the same patient on different days. d CSF, cerebrospinal fluid. I Four pneumococci from the same patient on different days. f Three specimens from the same patient on different days.
antigen was trapped in a glass tube, whereas it would be cleared by mucociliary action, phagocytosis, resorption, etc., in the human body. Pneumococci may also be cleared by being taken up into immune complexes (3, 13). On the other hand, the in vivo situation lends itself to constant replenishment of antigen from the lungs, where as much as 2 g of antigen may be present (8). Polysaccharide may also be released as pneumococci are digested by leukocytes (3). The number of pneumococci present (5, 7, 20) and the amount of antigen present (3, 4, 20) determine the prognosis of the patient. The number of microorganisms present may also tell us whether this is a clinical infection, as higher numbers of microorganisms have been found in patients with clinical infections than in carriers (21). Pneumococcal carriers with colds had pneumococci in concentrations below the TABLE 2. Minimum number of pneumococci required for LA Capsular type
3 5 6 7 9 10 11 13 14 18 19 22 23 31
Untyped
No. of pneumococci required
No. tested
Mean (per ml)
4 1 1 2 1 1 2 6 3 1 9 2 1 1 13
detection level for CIE (16). Patients with chronic bronchitis who are colonized with pneumococci in the lower respiratory tract even in the stable phases of their illness may pose a problem for the LA test, but while probable pathogens are present in high numbers (1, 17, 19, 23), carriers of potential pathogens probably have lower numbers of microorganisms than do patients who are clinically ill (21). In 11 patients with bronchitis from whom pneumococci were isolated, only 5 were antigen positive (24). Approximately 106 microorganisms per ml are required before a culture will yield colonies on an agar plate (17), and >105/ml are needed for microorganisms to be visible microscopically (5). In an article published in 1943, before the advent of penicillin, it was shown that the risk to the patient increases as the number of pneumococci present increases, with fatality rising from 2% when 76 are seen (7).
1.9 3.6 2.1 1.6 2.9 5.7 1.8 2.7 4.9 6.3 9.8 2.2 1.6 7.3 2.3
106 107 106 107 x 107
x x x x
x x x x x x x x x x
1.3 x 106-2.5 x 106 3.7 x 106-2.8 x 107
107 107 107 106
106 106 107 107
1.5 x 107-2.0 x 107 4.8 x 106-7.4 x 107 1.1 x 106-1.2 x 107 1.3 x 106-2.8 x 107 2.1 x 107-2.3 x 107
M
M
C R 0 o R G A N I s IA
Type 23
7
10 21 10
6
5
10 4 10 10
3 2
IA
10
L
10 0
24
46
72
0
24
48
72
TIME (HOURS)
106 107
Type 7
10 a
Range (per ml)
4.6 x 105-7.8 x 107
FIG. 2. Pneumococcal capsular antigen detection in BHI broth for 72 h. Symbols: 0, no antigen detectable; U, antigen detectable.
NOTES
VOL. 30, 1992
Purulent samples of sputum contain 105 to 108 pneumococci per ml (12), and the number of pneumococci in the sputa of pneumococcal pneumonia patients has been shown to be at least 106/ml (23). The point when Gram stain and culture are positive and at which the pneumococcus becomes a threat to the patient is also indicated when LA becomes positive, and it can be demonstrated within minutes on a sputum sample, which is easy to obtain. A further help in diagnosis is the fact that antigen remains positive even after the culture becomes negative (Fig. 2) and after antibiotic therapy (unpublished results). As antigen detection in sputum is not influenced by washing (11), we can also safely wash sputum specimens to make a culture more reliable. Pneumococci from pneumococcal pneumonia patients are not washed away (lla). LA probably provides us with the ability to determine the point at which detection of pneumococcal antigen becomes meaningful for the patient and is a good monitor for remaining antigen when the culture becomes negative. REFERENCES 1. Bartlett, J. G., and S. M. Finegold. 1978. Bacteriology of expectorated sputum with quantitative culture and wash technique compared to transtracheal aspirates. Am. Rev. Respir. Dis. 117:1019-1027. 2. Browne, K., J. Miegel, and K. D. Stottmeier. 1984. Detection of pneumococci in blood cultures by latex agglutination. J. Clin. Microbiol. 19:649-650. 3. Coonrod, J. D., and D. P. Drennan. 1976. Pneumococcal pneumonia: capsular polysaccharide antigenemia and antibody responses. Ann. Intern. Med. 84:254-260. 4. Dochez, A. R., and 0. T. Avery. 1917. The elaboration of specific soluble substance by pneumococcus during growth. J. Exp. Med. 26:477-493. 5. Feldman, W. E. 1977. Relation of concentrations of bacteria and of bacterial antigen in cerebrospinal fluid to prognosis in patients with bacterial meningitis. N. Engl. J. Med. 296:433-435. 6. Fossieck, B., and J. Fedorko. 1979. Counterimmunoelectrophoresis of blood cultures. Am. J. Clin. Pathol. 71:326-329. 7. Frisch, A. W., A. E. Price, and G. B. Myers. 1943. Pneumococcic pneumonia: the prognostic significance of the number of pneumococci in the sputum in relation to therapy, bacteremia, type, leucocyte count, duration of the disease, age, and degree of involvement. J. Clin. Invest. 22:207-214. 8. Frisch, A. W., J. T. Tripp, C. D. Barrett, and B. E. Pidgeon. 1942. The specific polysaccharide content of pneumonia lungs. J. Exp. Med. 76:505-510. 9. Fung, J. C., and K. Wicher. 1981. Minimum number of bacteria needed for antigen detection by counterimmunoelectrophoresis: in vivo and in vitro studies. J. Clin. Microbiol. 13:681-687. 10. Guzzetta, P., G. B. Toews, K. J. Robertson, and A. K. Pierce. 1983. Rapid diagnosis of community-acquired bacterial pneumonia. Am. Rev. Respir. Dis. 128:461-464. 11. Holloway, Y., W. G. Boersma, H. Kuttschrutter, and J. A. M.
519
Snijder. 1991. Effect of washing sputum on detection of pneumococcal capsular antigen. Eur. J. Clin. Microbiol. Infect. Dis. 10:567-569. 11a.Holloway, Y., W. G. Boersma, H. Kuttschrutter, and J. A. M. Snijder. Impact of washing sputum on streptococcus pneumoniae and other microorganisms. J. Infect. Dis., in press. 12. Kalin, M., A. A. Lindberg, and G. Tunevall. 1983. Etiological diagnosis of bacterial pneumonia by Gram stain and quantitative culture of expectorates. Scand. J. Infect. Dis. 15:153-160. 13. Kaplan, S. L. 1983. Antigen detection in cerebrospinal fluidpros and cons. Am. J. Med. (Infect. Dis. Symp.) 75(1B):109118. 14. Kenny, G. E., B. B. Wentworth, R. P. Beasley, and H. M. Foy. 1972. Correlation of circulating capsular polysaccharide with bacteremia in pneumococcal pneumonia. Infect. Immun. 6:431437. 15. Lenthe-Eboa, S., G. Brighouse, R. Auckenthaler, D. Lew, A. Zwahlen, P.-H. Lambert, and F. A. Waldvogel. 1987. Comparison of immunological methods for diagnosis of pneumococcal pneumonia in biological fluids. Eur. J. Clin. Microbiol. 6:28-34. 16. Miller, J., M. A. Sande, J. M. Gwaltney, and J. 0. Hendley. 1978. Diagnosis of pneumococcal pneumonia by antigen detection in sputum. J. Clin. Microbiol. 7:459-462. 17. Monroe, P. W., H. G. Muchmore, F. G. Felton, and J. K. Pirtle. 1969. Quantitation of microorganisms in sputum. Appl. Microbiol. 18:214-220. 18. Olcen, P. 1978. Serological methods for rapid diagnosis of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae in cerebrospinal fluid: a comparison of coagglutination, immunofluorescence and immunoelectroosmophoresis. Scand. J. Infect. Dis. 10:283-289. 19. Pirtle, J. K., P. W. Monroe, T. K. Smalley, J. A. Mohr, and E. R. Rhoades. 1969. Diagnostic and therapeutic advantages of serial quantitative cultures of fresh sputum in acute bacterial pneumonia. Am. Rev. Respir. Dis. 100:831-838. 20. Rytel, M. W., and L. C. Preheim. 1986. Antigen detection in the diagnosis and in the prognostic assessment of bacterial pneumonias. Diagn. Microbiol. Infect. Dis. 4:35S-46S. 21. S6derstrom, M., B. Hovelius, K. Prellner, and C. Schalen. 1990. Quantification of nasopharyngeal bacteria for diagnosis of respiratory tract infection in children. Scand. J. Infect. Dis. 22: 333-337. 22. Sottile, M. I., and M. W. Rytel. 1975. Application of counterimmunoelectrophoresis in the identification of Streptococcus pneumoniae in clinical isolates. J. Clin. Microbiol. 2:173-177. 23. Tebbutt, G. M., and D. J. Coleman. 1978. Evaluation of some methods for the laboratory examination of sputum. J. Clin. Pathol. 31:724-729. 24. Verhoef, J., and D. M. Jones. 1974. Pneumococcal antigen in sputum. Lancet i:879. 25. Whittle, H. C., P. Tugwell, L. J. Egler, and B. M. Greenwood. 1974. Rapid bacteriological diagnosis of pyogenic meningitis by latex agglutination. Lancet ii:619-621. 26. Yolken, R. H., D. Davis, J. Winkelstein, H. Russell, and J. E. Sippel. 1984. Enzyme immunoassay for detection of pneumococcal antigen in cerebrospinal fluid. J. Clin. Microbiol. 20:802805.
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1992, p. 517-519
0095-1137/92/020517-03$02.00/0 Copyright C 1992, American Society for Microbiology
Minimum Number of Pneumococci Required for Capsular Antigen To Be Detectable by Latex Agglutination YVETTE HOLLOWAY,'* WILLEM G. BOERSMA,2 HENRIETTE KUTTSCHRUTTER,l AND JAN A. M. SNIJDER1 Immunology Research, Regional Public Health Laboratory,' and Department of Pulmonary Diseases, University Hospital,2 Groningen, The Netherlands Received 29 July 1991/Accepted 5 November 1991
Forty-eight strains of Streptococcus pneumoniae were tested in vitro to determine the minimum number required for pneumococcal capsular antigen to be detectable by latex agglutination. It was found that 106 to 107 microorganisms per ml were needed and that antigen remained detectable even when viable pneumococci could no longer be demonstrated.
Pneumococcal capsular antigen has been detected by many methods, and all of these have a threshold of detection, with the lowest reported range running from 103/ml for enzyme-linked immunosorbent assay (26) and counterimmunoelectrophoresis (CIE; 13) and an average of 106/ml for these and other methods (2-4, 6, 9, 10, 15, 18, 22). In most instances, Omniserum (Staten Serum Institut, Copenhagen, Denmark) was used, but some workers, who obtained lower thresholds, used species-specific cell wall antiserum (26) or type-specific antiserum (9). With the latter, chances of detecting a single type of pneumococcus are better than they are with Omniserum (14). In order to establish the threshold number of pneumococci required for capsular antigen to be detectable by latex agglutination (LA), 48 pneumococci isolated from 35 patients were tested. The sources and types are listed in Table 1. Pneumococci were removed from blood agar plates with a cotton swab, and serial dilutions of 10-1 to 10' were prepared in physiological saline. These suspensions were divided into two parts: one to be used for antigen detection and one for quantitative cultures. Pneumococcal capsular antigen detection was performed by LA, using the Wellcogen Streptococcus pneumoniae Kit (Wellcome Diagnostics, Dartford, England) according to the manufacturer's instructions. The second part of the dilution row was used for estimation of the number of pneumococci present in each tube. Tubes were vortexed, and 25 ,ul from each tube was spread out evenly on a blood agar plate by using bent glass rods. The number of pneumococci per milliliter in each tube was calculated by multiplying the number of colonies grown by the dilution factor of that tube and then by the additional dilution factor of an inoculum of 25 RIu. For growth curves, three serial dilutions (10-3, 10', and 10-7 pneumococci per ml) were prepared in brain heart infusion (BHI) broth. Suspensions were vortexed, 500 p,l was removed from each tube for antigen detection, and 25 ,ud was removed for quantitative culture as described above. This process was repeated at 4, 8, 24, 48, and 72 h. BHI tubes were incubated at 37°C in 5% CO2 until growth curves were completed. It was found that approximately 106 to 107 pneumococci per ml were required for pneumococcal capsular antigen to
be detectable by LA directly from blood plates. Tubes containing fewer pneumococci per milliliter did not yield agglutination by LA. This number of pneumococci was required regardless of the source, the patient category, or the capsular type. Results for capsular types are listed in Table 2. In growth curves, pneumococcal capsular antigen could not be detected until the microorganisms had reached a concentration of approximately 106 to 107/ml in BHI broth. Typical examples are shown in Fig. 1. Once the concentration of 106 to 107 pneumococci per ml was reached, antigen remained detectable for the entire 72 h of the experiment, even when the number of pneumococci fell below this number or when no further viable pneumococci could be detected (Fig. 2). On the whole, CIE tends to react earlier than does LA, but this is probably because LA detects an antigen which is different from that detected in CIE (25). It is reported elsewhere that detectable levels of antigen are related to the logarithmic phase of growth (9), but we were unable to confirm this. Pneumococci well into the logarithmic phase (Fig. 1) still did not produce detectable antigen until the required number was attained, and microorganisms that were dying or dead (Fig. 2) still produced detectable antigen providing the threshold had previously been reached. An in vitro experiment such as ours cannot be entirely correlated to the in vivo situation because pneumococcal
10 m I C R
0 0 R G A N I S M S
10
10 10
Type 23 Type 7
a
Type 3
7
10 6
10 10
/
10
L
10
6
W-,o
4 3
0 2 4 6 *
Type 19
9
0 2 4 6 8 o0 2 4 TIME (HOURS)
0 2 4 6 8
FIG. 1. Pneumococcal capsular antigen detection in BHI broth. Symbols: 0, no antigen detectable; *, antigen detectable.
Corresponding author. 517
518
NOTES
J. CLIN. MICROBIOL. TABLE 1. Types and origins of 48 pneumococci from 35 patients No. of isolates
Source (n)
Category of patients (n)
3
4
5 6 7 9 10 11 13 14
1 1 2 1 1 2 6 3
18 19
1 9
Blood (1), throat (1), sputum (1) Blood (1) Sputum (1) Sputum (1) Blood (1), sputum (1) Blood (1) Sputum (1) Throat (1), sputum (1) Sputum (6) Blood (2) Sputum (1) CSFd (1) Bronchial washings (2), throat (1), sputum (3) Blood (1) Sputum (1) Sputum (1) Sputum (2) Throat (1) Sputum (1) Sputum (1) Nose (2), throat (8), saliva (2)
Pneumococcal pneumonia (2a) Pneumococcal meningitis (1) COPD with LRTIb (1) COPD with LRTI (1) Pneumococcal pneumonia (2) Pneumococcal pneumonia (1) COPD with LRTI (1) Pneumococcal pneumonia (1a) Pneumococcal pneumonia (1C) Pneumococcal pneumonia (2) COPD with LRTI (1) Pneumococcal meningitis (1) COPD with LRTI (3Y) Pneumococcal pneumonia (1) Pneumococcal pneumonia (1) LRTI (1) Pneumococcal pneumonia (1a) Pneumococcal pneumonia (1) Pneumococcal pneumonia (1) COPD with LRTI (1) COPD out-patients (10f)
Pneumococcal type
22 23 31 Untyped
2 1 1 13
a Two pneumococci from the same patient on different days. b COPD, chronic obstructive pulmonary disease; LRTI, lower respiratory tract infection. c Six sputum samples from the same patient on different days. d CSF, cerebrospinal fluid. I Four pneumococci from the same patient on different days. f Three specimens from the same patient on different days.
antigen was trapped in a glass tube, whereas it would be cleared by mucociliary action, phagocytosis, resorption, etc., in the human body. Pneumococci may also be cleared by being taken up into immune complexes (3, 13). On the other hand, the in vivo situation lends itself to constant replenishment of antigen from the lungs, where as much as 2 g of antigen may be present (8). Polysaccharide may also be released as pneumococci are digested by leukocytes (3). The number of pneumococci present (5, 7, 20) and the amount of antigen present (3, 4, 20) determine the prognosis of the patient. The number of microorganisms present may also tell us whether this is a clinical infection, as higher numbers of microorganisms have been found in patients with clinical infections than in carriers (21). Pneumococcal carriers with colds had pneumococci in concentrations below the TABLE 2. Minimum number of pneumococci required for LA Capsular type
3 5 6 7 9 10 11 13 14 18 19 22 23 31
Untyped
No. of pneumococci required
No. tested
Mean (per ml)
4 1 1 2 1 1 2 6 3 1 9 2 1 1 13
detection level for CIE (16). Patients with chronic bronchitis who are colonized with pneumococci in the lower respiratory tract even in the stable phases of their illness may pose a problem for the LA test, but while probable pathogens are present in high numbers (1, 17, 19, 23), carriers of potential pathogens probably have lower numbers of microorganisms than do patients who are clinically ill (21). In 11 patients with bronchitis from whom pneumococci were isolated, only 5 were antigen positive (24). Approximately 106 microorganisms per ml are required before a culture will yield colonies on an agar plate (17), and >105/ml are needed for microorganisms to be visible microscopically (5). In an article published in 1943, before the advent of penicillin, it was shown that the risk to the patient increases as the number of pneumococci present increases, with fatality rising from 2% when 76 are seen (7).
1.9 3.6 2.1 1.6 2.9 5.7 1.8 2.7 4.9 6.3 9.8 2.2 1.6 7.3 2.3
106 107 106 107 x 107
x x x x
x x x x x x x x x x
1.3 x 106-2.5 x 106 3.7 x 106-2.8 x 107
107 107 107 106
106 106 107 107
1.5 x 107-2.0 x 107 4.8 x 106-7.4 x 107 1.1 x 106-1.2 x 107 1.3 x 106-2.8 x 107 2.1 x 107-2.3 x 107
M
M
C R 0 o R G A N I s IA
Type 23
7
10 21 10
6
5
10 4 10 10
3 2
IA
10
L
10 0
24
46
72
0
24
48
72
TIME (HOURS)
106 107
Type 7
10 a
Range (per ml)
4.6 x 105-7.8 x 107
FIG. 2. Pneumococcal capsular antigen detection in BHI broth for 72 h. Symbols: 0, no antigen detectable; U, antigen detectable.
NOTES
VOL. 30, 1992
Purulent samples of sputum contain 105 to 108 pneumococci per ml (12), and the number of pneumococci in the sputa of pneumococcal pneumonia patients has been shown to be at least 106/ml (23). The point when Gram stain and culture are positive and at which the pneumococcus becomes a threat to the patient is also indicated when LA becomes positive, and it can be demonstrated within minutes on a sputum sample, which is easy to obtain. A further help in diagnosis is the fact that antigen remains positive even after the culture becomes negative (Fig. 2) and after antibiotic therapy (unpublished results). As antigen detection in sputum is not influenced by washing (11), we can also safely wash sputum specimens to make a culture more reliable. Pneumococci from pneumococcal pneumonia patients are not washed away (lla). LA probably provides us with the ability to determine the point at which detection of pneumococcal antigen becomes meaningful for the patient and is a good monitor for remaining antigen when the culture becomes negative. REFERENCES 1. Bartlett, J. G., and S. M. Finegold. 1978. Bacteriology of expectorated sputum with quantitative culture and wash technique compared to transtracheal aspirates. Am. Rev. Respir. Dis. 117:1019-1027. 2. Browne, K., J. Miegel, and K. D. Stottmeier. 1984. Detection of pneumococci in blood cultures by latex agglutination. J. Clin. Microbiol. 19:649-650. 3. Coonrod, J. D., and D. P. Drennan. 1976. Pneumococcal pneumonia: capsular polysaccharide antigenemia and antibody responses. Ann. Intern. Med. 84:254-260. 4. Dochez, A. R., and 0. T. Avery. 1917. The elaboration of specific soluble substance by pneumococcus during growth. J. Exp. Med. 26:477-493. 5. Feldman, W. E. 1977. Relation of concentrations of bacteria and of bacterial antigen in cerebrospinal fluid to prognosis in patients with bacterial meningitis. N. Engl. J. Med. 296:433-435. 6. Fossieck, B., and J. Fedorko. 1979. Counterimmunoelectrophoresis of blood cultures. Am. J. Clin. Pathol. 71:326-329. 7. Frisch, A. W., A. E. Price, and G. B. Myers. 1943. Pneumococcic pneumonia: the prognostic significance of the number of pneumococci in the sputum in relation to therapy, bacteremia, type, leucocyte count, duration of the disease, age, and degree of involvement. J. Clin. Invest. 22:207-214. 8. Frisch, A. W., J. T. Tripp, C. D. Barrett, and B. E. Pidgeon. 1942. The specific polysaccharide content of pneumonia lungs. J. Exp. Med. 76:505-510. 9. Fung, J. C., and K. Wicher. 1981. Minimum number of bacteria needed for antigen detection by counterimmunoelectrophoresis: in vivo and in vitro studies. J. Clin. Microbiol. 13:681-687. 10. Guzzetta, P., G. B. Toews, K. J. Robertson, and A. K. Pierce. 1983. Rapid diagnosis of community-acquired bacterial pneumonia. Am. Rev. Respir. Dis. 128:461-464. 11. Holloway, Y., W. G. Boersma, H. Kuttschrutter, and J. A. M.
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