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Demonstration of pneumolysin antibodies in circulating immune complexes—a new diagnostic method for pneumococcal pneumonia
Serodiugnosis
and Immunotherapy
in Infectious Disease (1990)4, 451458
Demonstration of pneumolysin antibodies in circulating immune complexes- a new diagnostic method for pneumococcal pneumonia
Maija Leinonen I*, Hannu SyrjW, Erja Jalonen’, Pekka Kujala3 and Elja Herva’ ‘National Public Health Institute, Mannerheimintie 166 SF-00300 Helsinki, Finland, ‘National Public Health Institute, Oulu, Finland and 3Department of Medicine, Universit? Central Hospital, Oulu. Finland
We isolatedimmunecomplexes(IC) from the seraobtainedfrom 129adult patients with community-acquiredpneumoniaand from 120healthy controls and measured antibodiesto pneumococcalpneumolysinin dissociatedcomplexes.None of the healthy controls had demonstrablepneumolysin-specific IC in their serairrespective of whetherthey had high or low serumantibody titers to pneumolysin.By contrast, 62 (48%) of pneumoniapatients had pneumolysin-ICin one or more of serum specimens. All 1I patientswith positiveblood culture for pneumococcus and 86% of those in whom pneumococcalinvolvement was suggestedby culture or antigen detection in sputumor who showedserumantibody responseto pneumolysinhad pneumolysinantibodiesin circulating IC. In addition, pneumolysin-ICweredetected in 17 patients without other evidence of pneumococcalinfection. The lack of pneumolysin-ICin healthy controlsand their presencein patientswith pneumococcal pneumoniasuggestthat the demonstrationpneumolysin-ICis a sensitive,reliable methodfor the diagnosisof pneumococcalpneumonia. Keywords: Immunecomplexes,pneumolysin,pneumonia.
Introduction Recently, an enzyme immunoassay (EIA) for measuring antibodies to pneumococcal protein toxin, pneumolysin, has been developed and applied to the etiological diagnosis of pneumonia in different patient material?. The pneumolysin EIA has proven to be a sensitive method compared to both culture and antigen detection methods as well as to antibody assaysfor pneumococcal polysaccharideG4. Nevertheless, someblood culturepositive patients have remained negative by this assay. When considering reasons for this we thought it possible that antibody responsescould be masked by the antibodies forming immune complexes with circulating pneumolysin. To test this possibility we isolated immune complexes (IC) from the acute and convalescent sera of 129 patients with community-acquired pneumonia, and asked whether they would contain antibodies to pneumolysin. The findings gave strong support to the hypothesis: pneumolysinPreliminary report of this study was presented at the 30th ICAAC * Author to whom correspondence should be addressed.
meeting
in Atlanta,
October
21-25.
1990.
451 088&0786/90/060451+08
$03.00/O
0 1990AcademicPressLimited
452
M. Leinonen
et
al.
specific IC were found in each of I I blood culture-positive patients and in 86% of patients with pneumococci in sputum samples. As a whole, our results suggest that demonstration of these IC may be the most sensitive diagnostic test for pneumococcal pneumonia. Materials and methods Patients and specimens
The patient material consisted of 129 consecutive, adult patients (mean age 60.6 years, range 15 to 93 years) with X-ray positive community-acquired pneumonia admitted to Oulu University Central Hospital between May 6, 1986 and May 5, 1987 and from whom at least two serum samples were available. Blood cultures were obtained from all patients on admission. Sputum samples were available from 111 patients and they were usually taken before the antibiotic treatment was started. Only the sputa taken before noon were tested during the same working day; the specimens taken during nights or weekends were stored at +4”C until studied. Acute serum samples were obtained on admission from all the 129 patients. A second serum sample, taken 5-8 days later, when the patient was discharged from hospital, was available from 94 patients and a third serum sample, taken during the follow-up visit 4-5 weeks after admission, was obtained from 58 patients. Control sera
Sera from 109 healthy children (6 months to 18 years old) and from 11 healthy adults (age 20 to 30 years), containing both high and low antibody titers to pneumolysin, were used as controls in immune complex studies. Sputum studies
Sputum samples were treated with the same volume of N-acetyl-L-cysteine solution and cultured on blood and chocolate agar plates. Pneumococcal antigens were demonstrated by the latex agglutination test’ after boiling the treated sputa using latex reagent prepared from pneumococcal Omniserum (Statens Serum Institute, Copenhagen, Denmark). Isolation and dissociation of immune complexes (IC)
Immune complexes were isolated by slightly modifying the method described in detail previously6. Briefly, for precipitation of ICs 100 pl of 7% polyethyleneglycol (PEG 6000) in borate buffer, pH 8.2 was added to 100 pl of serum and the mixture was incubated overnight at +4”C. Precipitated complexes were washed twice with 3.5% PEG after which they were dissociated with 100 pl of borate buffer, pH 12.0. Measurement of antibodies to pneumococcalpneumolysin
IgG antibodies to pneumolysin were measured by EIA as described in detail previously3. Briefly, 96-well EIA plates (NUNC Maxisorb, Roskilde, Denmark) were coated with pneumolysin in phosphate-buffered saline, pH 7.4 (PBS) at + 37°C overnight and postcoated for 1 h with 5% sheep serum. Sera were diluted l/100, l/l000 and l/10,000 and
453
Pneumolysin immune complexes in pneumonia
dissociated complexes l/SO, l/500 and l/5000 with PBS containing 10% fetal calf serum (PBS-FCS) and incubated in the wells at + 37°C for 2 h. After incubation with conjugate, alkaline phosphatase conjugated anti-human IgG (Orion Diagnostics, Espoo, Finland), diluted with PBS-FCS, the substrate, 1 mg of paranitrophenylphosphate (Sigma 104) in 1 ml of diethanolamine buffer, pH 96 was added and incubated for 30 min at + 37°C. Optical density values were read at 405 nm by Multiscan spectrophotometer (Ef-Lab, Finland). Antibody titers were expressed as highest dilution giving an OD value of 0.15. Diagnostic
criteria,for
pneumococcal
(Pnc) etiolog?
Isolation of Pnc from blood culture was considered as a definite etiological diagnosis. The demonstration of Pnc in sputum by culture or antigen detection was considered as suggestive for Pnc etiology. The diagnostic criterium for pneumolysin antibody assay was a two-fold or greater increase in antibody titer between paired sera; this criterium is based on results of paired sera from healthy adults?. ICs were considered to be pneumolysin-specific, if antibodies to pneumolysin were detected in the dissociated complexes in a titer higher than 1:lOO. When calculating sensitivity, specificity, positive and negative predictive values and efficiency’ for the diagnostic tests a true-positive Pnc finding was defined as one indicated by at least two of the diagnostic tests employed. Results
Table 1 shows that pneumolysin-IC were found in the sera of all 11 patients with positive blood culture for Pnc. The geometric mean antibody titers (GMT) of pneumolysin antibodies in the sera and in dissociated IC were close to each other; this was true both for the acute and two convalescent phase sera. By contrast, pneumolysin-IC were not found in any of the 120 healthy controls
pneumolysin
irrespective
of whether
they had high or low
antibody titers.
Table 1. Geometric mean titers (GMT) for pneumolysin antibodies in sera (Ply-ab) and in dissociated immune complexes (Ply-K) and presence of Ply-IC in 11 patients with positive blood culture for pneumococcus and 120 healthy controls divided into three age groups Ply-K Group Patients
Healthy controls
Serum sample
No. of sera
day 0 1 week 1 month
II 11 8
Age group <2Y 7-18 y >2oy
___~ 96 11 13
Ply-ab GMT
GMT
No. (%) present
276.4 916.8 650.0
200.9 566.4 567.1
8 (72.7) 10 (90.9) 8 (100.0)
253.1 3370.9 3743.8
~~~~-0 0 0
454
M. Leinonen et al.
A serum antibody response to pneumolysin was demonstrable in eight (72.7%) of the 11 blood culture-positive patients. Figure 1 shows the pneumolysin antibody titers in the sera and in dissociated IC in those eight blood culture-positive patients from whom three serum samples were available. In the patients (A, B and C) who did not show a serum antibody response to pneumolysin, antibodies were present in IC in all three sera, often in high titers. The antibody response was also relatively low in the patients E, F and G, in whom IC were present in high titers. especially in the second serum taken 1 week after the acute serum. Table 2 compares the detection of pneumolysin-IC to other diagnostic methods for Pnc etiology, blood culture, the demonstration of Pnc in sputum by culture or antigen detection and pneumolysin antibody assay. Pneumolysin-IC were found in 86% of those
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no.
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Figure 1. EIA antibody titers to pneumolysin in the sera (O-O) and in the dissociated immune complexes (M) of eight pneumonia patients with positive blood culture for pneumococcus and from whom three serum samples taken in acute phase (serum 0), 1 week (serum 1) and 1 month (serum 3) after admission were available.
455
Pneumolysin immune complexes in pneumonia
Table 2. Comparison of four diagnostic methods, blood culture (BC), detection of Pnc in sputum (SP Pnc), pneumolysin antibody assay (Ply-ab) and detection of pneumolysin antibodies in immune complexes (Ply-IC) in the etiological diagnosis of Pnc pneumonia No. (X) of positive findings by
Diagnostic method
No. of findings
BC
BC
11
11
SP Pnc
42 29 62
8 (19.0) 8 (27.5) 11 (17.4)
Ply-ab Ply-K
SP Pnc
Ply-ab
Ply-K
8 (72.7) 42 18 (62.1) 36 (58.1)
8 (72.7) 18 (42.8) 29 25 (40.3)
II (100.0) 36 (85.7) 25 (86.2) 62
diagnosed either by pneumolysin antibody assay or by detection of Pnc in sputum, whereas among the 62 IC-positive patients antibody response to pneumolysin was seen in only 40%, Pnc were demonstrated in sputum in 58% and blood culture was positive in 17%. In general, antibody response to pneumolysin was positive in 40% to 72%, Pnc was detected in sputum in 58% to 73% and blood culture was positive in 17% to 28% of patients with Pnc indicated by another diagnostic method. The sensitivities, specificities, positive and negative predictive values and efficiencies of the different diagnostic tests are shown in Table 3, based on defining a true positive result as one in which at least two of the tests give a concordant positive finding for Pnc. The sensitivity of IC demonstration was clearly highest, 98%, whereas the sensitivity of blood culture was only 24%. The specificity of IC detection was 83%, and that of pneumolysin antibody assay and detection of Pnc in sputum 97% and 94%, respectively. Positive and negative predictive values for IC detection were 73% and 99%, and for blood culture 100% and 74%, respectively. Two other diagnostic methods for Pnc, antibody assay and detection of Pnc in sputum, gave intermediate values. The test efficiency calculations indicated the highest value (99O/,) for IC detection, intermediate values of 85% and 93% for pneumolysin antibody assay and detection of Pnc in sputum and the lowest value, 73%, for blood culture. Table 4 shows IC findings in acute and two convalescent sera of patients in whom Pnc was demonstrated by other diagnostic methods. IC was detected in 59.2% of the acute
Table 3. Sensitivity @ENS), specificity (SPEC), positive (PPV) and negative (NPV) predictive values and efficiency (EFF) of different diagnositic tests, blood culture (BC), detection of Pnc in sputum (SP Pnc), antibody assay to pneumolysin (Ply-ab) and detection of pneumolysin immune complexes (Ply-IC) for Pnc in 129 pneumonia patients. True-positive Pnc finding was defined as one indicated by at least two of the diagnostic tests employed Diagnostic method ~-. ~~ BC SP Pnc Ply-ab Ply-IC
No. of findings
Positive only by this test
SENS %
SPEC %
PPV %
NPV %
EFF
11 37 ‘29 62
0 5 3 17
23.9 80.4 56.5 97.8
100.0 94.3 96.5 83.4
100.0 88.8 89.6 72.6
73.5 90.2 80.5 98.8
72.9 93.0 84.5 99.2
%
M. Leinonenet al.
456
Table4. The presence of pneumolysin immune complexes (Ply-IC) and the geometric mean titers (GMT) and range in acute (day 0), second (day 7) and third serum (day 40) of patients with Pnc pneumonia diagnosed by different methods, blood culture, demonstration of Pnc in sputum, demonstration of the antibody response to pneumolysin (Ply-response) and demonstration of pneumolysin immune complexes (Ply-IC) No. (%) of Ply-IC positive/no. GMT (range) Diagnostic
method
Blood culture Pnc in sputum* Ply-response? Ply-X$
day 0
day 7
8/ 11 (72.7) 201 (< lOtK-360) 20/34 (58.8) 232 (< lOtKlO8O) 3/9 (33.3) 118 (<100-460) IO/l7 (58.8) 167 ( < 1OCrSOO)
IO/l 1 (90.9) 566 (< 100-1230) 20/30 (66.7) 420 ( < 100-2090) S/S (62.5) 168 (< 100-530) 13/16 (81.5) 435 ( < 100-2220)
studied day 40 717 (100.0) 567 (<21&1050) 1 l/20 (55.0) 135 ( < 100-430) l/2 (50.0) 150 (-=z 10&170) 5/S (62.5) 213 (< lOCr500)
* Blood culture-positive cases excluded. t Sputum and blood culture-positive cases excluded. $ Cases positive with other methods excluded.
phase sera, in 72.7% of the sera taken 1 week later and in 63.9% of the sera taken 4-5 weeks after admission in those patients who had any evidence by any method of Pnc involvement. If only the first two sera taken I week apart are considered, pneumolysinIC were found in 89% of all IC-positive cases. In all diagnostic groups, IC antibody titers were highest in the second serum. The highest IC titers were seen in the blood culturepositive patients and the lowest titers in those patients in whom an antibody response to pneumolysin was the indication of Pnc involvement. All eight blood culture-positive patients from whom a third serum was available had IC in relatively high titers still present in the third serum (see also Figure l), whereas the IC titers in the other diagnostic groups were clearly lower in the third than in the second serum. Discussion
We could demonstrate the presence of circulating immune complexes containing pneumolysin antibodies in all 11 blood culture-positive pneumonia patients, three of which did not show an antibody response to pneumolysin. This finding confirms the hypothesis that led to this study: the relatively low sensitivity of pneumolysin antibody determination is due to the binding of pre-existing and newly formed antibodies to pneumolysin forming circulating IC. Altogether pneumolysin-IC were found in 62 (48%) of 129 patients which is in accordance with several studies that have shown the incidence of about 50% for Pnc as a cause of community-acquired pneumonia*-“. The sensitivity of the demonstration of pneumolysin antibodies in dissociated IC was shown to be very high, 98% compared to sensitivities of 24%, 57% and 84% of the other diagnostic methods, blood culture, pneumolysin antibody assay and detection of Pnc in sputum, respectively. Furthermore, the negative predictive value of 99% for IC detection
Pueumolysin immune complexes in pneumonia
457
indicates it to be a very efficient test for exclusion of Pnc involvement in pneumonia patients. The positive predictive value, however, was relatively low, 73%, because pneumolysin-IC was the most sensitive of the diagnostic methods. It is possible that the IC findings in the 17 patients without a confirmatory test are false-positive reactions or that the lack of a confirmatory finding is only due to the poor diagnostic efficiency of the other methods. Several lines of evidence support the hypothesis that the presence of pneumolysin-IC is diagnostic for Pnc pneumonia. We could not detect pneumolysin antibodies in IC isolated from 120 sera of healthy persons even though some of these sera contained free pneumolysin antibodies in very high titers (around 1/lO,OOO). In addition, false-positive IC findings due to carriage of Pnc in the upper respiratory tract can possibly be excluded by the fact that the carriage rate for Pnc in Finnish children is around 20% (unpublished data) and the 98 control sera obtained from children were all negative for pneumolysinIC. These findings suggest that the presence of pneumolysin-IC is a strong indication of Pnc infection. Pneumolysin is an intracellular protein toxin of Pnc and it is liberated from Pnc cells after their lysis I2. We can thus speculate that in bacteremic infections or in lung infections, when bacterial products have an easy access to circulation, IC containing pneumolysin and its antibodies are easily formed. When Pnc was indicated by any of the diagnostic methods pneumolysin-IC were present in 57% of the acute sera and in 83% of the paired sera taken 5-8 days apart. These data suggest that the detection of IC in acute serum samples could be used as a rapid diagnostic method. Although sputum studies might provide etiological diagnosis even more rapidly than the demonstration of pneumolysin-IC, by which the result can be obtained only on the following day, sputum specimens are not obtained from all patients and especially often not from those with the most severe infection. The handling and treatment of sputum samples is also laborious and time-consuming. The second advantage of IC determinations, especially when compared to normal antibody assays, is that paired sera taken not more than 1 week apart can be used for these studies and thus no extra hospital visit for convalescent serum is needed. In addition, it allows the specific antimicrobial therapy for most of the patients with Pnc pneumonia. In conclusion, the data presented strongly suggest that detection of pneumolysin antibodies in circulating immune complexes is a sensitive, reliable diagnostic method for pneumococcal pneumonia that can also be used as a rapid diagnostic test. The detection of specific antibodies from circulating immune complexes, earlier shown to be useful in Lyme disease6 and now in pneumococcal pneumonia, gives a new perspective for the etiological diagnosis of severe bacterial infections. Acknowledgements
We are grateful to Teija Jaakkola RN, Raili Kalliokokoski RN and Kaija Vilen RN for their skilful technical assistance. These studies have been partly supported by the Medical Council, Academy of Finland. References 1. Kahn M, Kanclerski K, Granstriim M, Mijllby R. Diagnosis of pneumococcal pneumonia by enzyme-linked immunosorbent assay of antibodies to pneumococcal hemolysin. J Ciin Microbial 1987; 25: 2269.
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2. Kanclerski K, Blomqvist S, Granstrom M, Mijllby R. Serumantibodiesto pneumolysinin patientswith pneumonia.J Clin Microbial 1988;26: 96-100. 3. Jalonen E, Paton JC, Koskela M, Kerttula Y, Leinonen M. Measurementof antibody responseto pneumolysin-a promisingmethod for the presumptiveetiological diagnosisof pneumococcalpneumonia.J Infect 1989;19: 127-34. 4. Ortqvist A, Grillner L, Hedlund J et al. Outcome and prognostic factors in communityacquiredpneumonia.Eur RespJ 1990;3: 1105-13. 5. LeinonenM. Detection of pneumococcalcapsularpolysaccharideantigensby latex agglutination, counterimmunoelectrophoresis and radioimmunoassayin middle ear exudatesin acute otitis media.J Clin Microbial 1980;11: 135-40. 6. Schutzer SE, Coyle PK, BelmanA, Golightly MG, Drulle J. Sequestrationof antibody to Borrelia burgdorferi in immunecomplexesin seronegativeLyme disease. Lancet 1990;i: 31215. 7. CohenRS. Useof predictive value theory in clinical immunology.In: RoseNR, FriedmanH, Fahey JL, eds. Manual of Clinical Laboratory Immunology. Washington D.C.: American Society for Microbiology, 1986:96670. 8. MacFarlane JT, Ward MJ, Finch RG, Macrae D. Hospital study of adult communityacquiredpneumonia.Lancet 1982;ii: 255-8. 9. Berntsson E, Blomberg J, Lagergard T, Trollfors B. Etiology of community-acquired pneumoniain patientsrequiring hospitalization, Eur J Clin Microbial 1985;4: 268-72. 10. HolmbergH. Etiology of community-acquiredpneumoniain hospitaltreated patients.Stand J Inf Dis 1987;19: 491-501. 1I. Marrie TJ, Durant H, Yates L. Community-acquiredpneumoniarequiring hospitalization. Rev Inf Dis 1989;11: 586-99. 12. Berry AM, Lock RA, HansmanD, Paton JC. Contribution of autolysin to virulence of Streptococcus pneumoniae. Infect Immunol 1989;57: 232430. (Manuscript accepted 3 May 1991)
and Immunotherapy
in Infectious Disease (1990)4, 451458
Demonstration of pneumolysin antibodies in circulating immune complexes- a new diagnostic method for pneumococcal pneumonia
Maija Leinonen I*, Hannu SyrjW, Erja Jalonen’, Pekka Kujala3 and Elja Herva’ ‘National Public Health Institute, Mannerheimintie 166 SF-00300 Helsinki, Finland, ‘National Public Health Institute, Oulu, Finland and 3Department of Medicine, Universit? Central Hospital, Oulu. Finland
We isolatedimmunecomplexes(IC) from the seraobtainedfrom 129adult patients with community-acquiredpneumoniaand from 120healthy controls and measured antibodiesto pneumococcalpneumolysinin dissociatedcomplexes.None of the healthy controls had demonstrablepneumolysin-specific IC in their serairrespective of whetherthey had high or low serumantibody titers to pneumolysin.By contrast, 62 (48%) of pneumoniapatients had pneumolysin-ICin one or more of serum specimens. All 1I patientswith positiveblood culture for pneumococcus and 86% of those in whom pneumococcalinvolvement was suggestedby culture or antigen detection in sputumor who showedserumantibody responseto pneumolysinhad pneumolysinantibodiesin circulating IC. In addition, pneumolysin-ICweredetected in 17 patients without other evidence of pneumococcalinfection. The lack of pneumolysin-ICin healthy controlsand their presencein patientswith pneumococcal pneumoniasuggestthat the demonstrationpneumolysin-ICis a sensitive,reliable methodfor the diagnosisof pneumococcalpneumonia. Keywords: Immunecomplexes,pneumolysin,pneumonia.
Introduction Recently, an enzyme immunoassay (EIA) for measuring antibodies to pneumococcal protein toxin, pneumolysin, has been developed and applied to the etiological diagnosis of pneumonia in different patient material?. The pneumolysin EIA has proven to be a sensitive method compared to both culture and antigen detection methods as well as to antibody assaysfor pneumococcal polysaccharideG4. Nevertheless, someblood culturepositive patients have remained negative by this assay. When considering reasons for this we thought it possible that antibody responsescould be masked by the antibodies forming immune complexes with circulating pneumolysin. To test this possibility we isolated immune complexes (IC) from the acute and convalescent sera of 129 patients with community-acquired pneumonia, and asked whether they would contain antibodies to pneumolysin. The findings gave strong support to the hypothesis: pneumolysinPreliminary report of this study was presented at the 30th ICAAC * Author to whom correspondence should be addressed.
meeting
in Atlanta,
October
21-25.
1990.
451 088&0786/90/060451+08
$03.00/O
0 1990AcademicPressLimited
452
M. Leinonen
et
al.
specific IC were found in each of I I blood culture-positive patients and in 86% of patients with pneumococci in sputum samples. As a whole, our results suggest that demonstration of these IC may be the most sensitive diagnostic test for pneumococcal pneumonia. Materials and methods Patients and specimens
The patient material consisted of 129 consecutive, adult patients (mean age 60.6 years, range 15 to 93 years) with X-ray positive community-acquired pneumonia admitted to Oulu University Central Hospital between May 6, 1986 and May 5, 1987 and from whom at least two serum samples were available. Blood cultures were obtained from all patients on admission. Sputum samples were available from 111 patients and they were usually taken before the antibiotic treatment was started. Only the sputa taken before noon were tested during the same working day; the specimens taken during nights or weekends were stored at +4”C until studied. Acute serum samples were obtained on admission from all the 129 patients. A second serum sample, taken 5-8 days later, when the patient was discharged from hospital, was available from 94 patients and a third serum sample, taken during the follow-up visit 4-5 weeks after admission, was obtained from 58 patients. Control sera
Sera from 109 healthy children (6 months to 18 years old) and from 11 healthy adults (age 20 to 30 years), containing both high and low antibody titers to pneumolysin, were used as controls in immune complex studies. Sputum studies
Sputum samples were treated with the same volume of N-acetyl-L-cysteine solution and cultured on blood and chocolate agar plates. Pneumococcal antigens were demonstrated by the latex agglutination test’ after boiling the treated sputa using latex reagent prepared from pneumococcal Omniserum (Statens Serum Institute, Copenhagen, Denmark). Isolation and dissociation of immune complexes (IC)
Immune complexes were isolated by slightly modifying the method described in detail previously6. Briefly, for precipitation of ICs 100 pl of 7% polyethyleneglycol (PEG 6000) in borate buffer, pH 8.2 was added to 100 pl of serum and the mixture was incubated overnight at +4”C. Precipitated complexes were washed twice with 3.5% PEG after which they were dissociated with 100 pl of borate buffer, pH 12.0. Measurement of antibodies to pneumococcalpneumolysin
IgG antibodies to pneumolysin were measured by EIA as described in detail previously3. Briefly, 96-well EIA plates (NUNC Maxisorb, Roskilde, Denmark) were coated with pneumolysin in phosphate-buffered saline, pH 7.4 (PBS) at + 37°C overnight and postcoated for 1 h with 5% sheep serum. Sera were diluted l/100, l/l000 and l/10,000 and
453
Pneumolysin immune complexes in pneumonia
dissociated complexes l/SO, l/500 and l/5000 with PBS containing 10% fetal calf serum (PBS-FCS) and incubated in the wells at + 37°C for 2 h. After incubation with conjugate, alkaline phosphatase conjugated anti-human IgG (Orion Diagnostics, Espoo, Finland), diluted with PBS-FCS, the substrate, 1 mg of paranitrophenylphosphate (Sigma 104) in 1 ml of diethanolamine buffer, pH 96 was added and incubated for 30 min at + 37°C. Optical density values were read at 405 nm by Multiscan spectrophotometer (Ef-Lab, Finland). Antibody titers were expressed as highest dilution giving an OD value of 0.15. Diagnostic
criteria,for
pneumococcal
(Pnc) etiolog?
Isolation of Pnc from blood culture was considered as a definite etiological diagnosis. The demonstration of Pnc in sputum by culture or antigen detection was considered as suggestive for Pnc etiology. The diagnostic criterium for pneumolysin antibody assay was a two-fold or greater increase in antibody titer between paired sera; this criterium is based on results of paired sera from healthy adults?. ICs were considered to be pneumolysin-specific, if antibodies to pneumolysin were detected in the dissociated complexes in a titer higher than 1:lOO. When calculating sensitivity, specificity, positive and negative predictive values and efficiency’ for the diagnostic tests a true-positive Pnc finding was defined as one indicated by at least two of the diagnostic tests employed. Results
Table 1 shows that pneumolysin-IC were found in the sera of all 11 patients with positive blood culture for Pnc. The geometric mean antibody titers (GMT) of pneumolysin antibodies in the sera and in dissociated IC were close to each other; this was true both for the acute and two convalescent phase sera. By contrast, pneumolysin-IC were not found in any of the 120 healthy controls
pneumolysin
irrespective
of whether
they had high or low
antibody titers.
Table 1. Geometric mean titers (GMT) for pneumolysin antibodies in sera (Ply-ab) and in dissociated immune complexes (Ply-K) and presence of Ply-IC in 11 patients with positive blood culture for pneumococcus and 120 healthy controls divided into three age groups Ply-K Group Patients
Healthy controls
Serum sample
No. of sera
day 0 1 week 1 month
II 11 8
Age group <2Y 7-18 y >2oy
___~ 96 11 13
Ply-ab GMT
GMT
No. (%) present
276.4 916.8 650.0
200.9 566.4 567.1
8 (72.7) 10 (90.9) 8 (100.0)
253.1 3370.9 3743.8
~~~~-0 0 0
454
M. Leinonen et al.
A serum antibody response to pneumolysin was demonstrable in eight (72.7%) of the 11 blood culture-positive patients. Figure 1 shows the pneumolysin antibody titers in the sera and in dissociated IC in those eight blood culture-positive patients from whom three serum samples were available. In the patients (A, B and C) who did not show a serum antibody response to pneumolysin, antibodies were present in IC in all three sera, often in high titers. The antibody response was also relatively low in the patients E, F and G, in whom IC were present in high titers. especially in the second serum taken 1 week after the acute serum. Table 2 compares the detection of pneumolysin-IC to other diagnostic methods for Pnc etiology, blood culture, the demonstration of Pnc in sputum by culture or antigen detection and pneumolysin antibody assay. Pneumolysin-IC were found in 86% of those
-I
D
1000 A t
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0
I
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B
-Al
I
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no.
1 I
1 2
Fk-
Figure 1. EIA antibody titers to pneumolysin in the sera (O-O) and in the dissociated immune complexes (M) of eight pneumonia patients with positive blood culture for pneumococcus and from whom three serum samples taken in acute phase (serum 0), 1 week (serum 1) and 1 month (serum 3) after admission were available.
455
Pneumolysin immune complexes in pneumonia
Table 2. Comparison of four diagnostic methods, blood culture (BC), detection of Pnc in sputum (SP Pnc), pneumolysin antibody assay (Ply-ab) and detection of pneumolysin antibodies in immune complexes (Ply-IC) in the etiological diagnosis of Pnc pneumonia No. (X) of positive findings by
Diagnostic method
No. of findings
BC
BC
11
11
SP Pnc
42 29 62
8 (19.0) 8 (27.5) 11 (17.4)
Ply-ab Ply-K
SP Pnc
Ply-ab
Ply-K
8 (72.7) 42 18 (62.1) 36 (58.1)
8 (72.7) 18 (42.8) 29 25 (40.3)
II (100.0) 36 (85.7) 25 (86.2) 62
diagnosed either by pneumolysin antibody assay or by detection of Pnc in sputum, whereas among the 62 IC-positive patients antibody response to pneumolysin was seen in only 40%, Pnc were demonstrated in sputum in 58% and blood culture was positive in 17%. In general, antibody response to pneumolysin was positive in 40% to 72%, Pnc was detected in sputum in 58% to 73% and blood culture was positive in 17% to 28% of patients with Pnc indicated by another diagnostic method. The sensitivities, specificities, positive and negative predictive values and efficiencies of the different diagnostic tests are shown in Table 3, based on defining a true positive result as one in which at least two of the tests give a concordant positive finding for Pnc. The sensitivity of IC demonstration was clearly highest, 98%, whereas the sensitivity of blood culture was only 24%. The specificity of IC detection was 83%, and that of pneumolysin antibody assay and detection of Pnc in sputum 97% and 94%, respectively. Positive and negative predictive values for IC detection were 73% and 99%, and for blood culture 100% and 74%, respectively. Two other diagnostic methods for Pnc, antibody assay and detection of Pnc in sputum, gave intermediate values. The test efficiency calculations indicated the highest value (99O/,) for IC detection, intermediate values of 85% and 93% for pneumolysin antibody assay and detection of Pnc in sputum and the lowest value, 73%, for blood culture. Table 4 shows IC findings in acute and two convalescent sera of patients in whom Pnc was demonstrated by other diagnostic methods. IC was detected in 59.2% of the acute
Table 3. Sensitivity @ENS), specificity (SPEC), positive (PPV) and negative (NPV) predictive values and efficiency (EFF) of different diagnositic tests, blood culture (BC), detection of Pnc in sputum (SP Pnc), antibody assay to pneumolysin (Ply-ab) and detection of pneumolysin immune complexes (Ply-IC) for Pnc in 129 pneumonia patients. True-positive Pnc finding was defined as one indicated by at least two of the diagnostic tests employed Diagnostic method ~-. ~~ BC SP Pnc Ply-ab Ply-IC
No. of findings
Positive only by this test
SENS %
SPEC %
PPV %
NPV %
EFF
11 37 ‘29 62
0 5 3 17
23.9 80.4 56.5 97.8
100.0 94.3 96.5 83.4
100.0 88.8 89.6 72.6
73.5 90.2 80.5 98.8
72.9 93.0 84.5 99.2
%
M. Leinonenet al.
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Table4. The presence of pneumolysin immune complexes (Ply-IC) and the geometric mean titers (GMT) and range in acute (day 0), second (day 7) and third serum (day 40) of patients with Pnc pneumonia diagnosed by different methods, blood culture, demonstration of Pnc in sputum, demonstration of the antibody response to pneumolysin (Ply-response) and demonstration of pneumolysin immune complexes (Ply-IC) No. (%) of Ply-IC positive/no. GMT (range) Diagnostic
method
Blood culture Pnc in sputum* Ply-response? Ply-X$
day 0
day 7
8/ 11 (72.7) 201 (< lOtK-360) 20/34 (58.8) 232 (< lOtKlO8O) 3/9 (33.3) 118 (<100-460) IO/l7 (58.8) 167 ( < 1OCrSOO)
IO/l 1 (90.9) 566 (< 100-1230) 20/30 (66.7) 420 ( < 100-2090) S/S (62.5) 168 (< 100-530) 13/16 (81.5) 435 ( < 100-2220)
studied day 40 717 (100.0) 567 (<21&1050) 1 l/20 (55.0) 135 ( < 100-430) l/2 (50.0) 150 (-=z 10&170) 5/S (62.5) 213 (< lOCr500)
* Blood culture-positive cases excluded. t Sputum and blood culture-positive cases excluded. $ Cases positive with other methods excluded.
phase sera, in 72.7% of the sera taken 1 week later and in 63.9% of the sera taken 4-5 weeks after admission in those patients who had any evidence by any method of Pnc involvement. If only the first two sera taken I week apart are considered, pneumolysinIC were found in 89% of all IC-positive cases. In all diagnostic groups, IC antibody titers were highest in the second serum. The highest IC titers were seen in the blood culturepositive patients and the lowest titers in those patients in whom an antibody response to pneumolysin was the indication of Pnc involvement. All eight blood culture-positive patients from whom a third serum was available had IC in relatively high titers still present in the third serum (see also Figure l), whereas the IC titers in the other diagnostic groups were clearly lower in the third than in the second serum. Discussion
We could demonstrate the presence of circulating immune complexes containing pneumolysin antibodies in all 11 blood culture-positive pneumonia patients, three of which did not show an antibody response to pneumolysin. This finding confirms the hypothesis that led to this study: the relatively low sensitivity of pneumolysin antibody determination is due to the binding of pre-existing and newly formed antibodies to pneumolysin forming circulating IC. Altogether pneumolysin-IC were found in 62 (48%) of 129 patients which is in accordance with several studies that have shown the incidence of about 50% for Pnc as a cause of community-acquired pneumonia*-“. The sensitivity of the demonstration of pneumolysin antibodies in dissociated IC was shown to be very high, 98% compared to sensitivities of 24%, 57% and 84% of the other diagnostic methods, blood culture, pneumolysin antibody assay and detection of Pnc in sputum, respectively. Furthermore, the negative predictive value of 99% for IC detection
Pueumolysin immune complexes in pneumonia
457
indicates it to be a very efficient test for exclusion of Pnc involvement in pneumonia patients. The positive predictive value, however, was relatively low, 73%, because pneumolysin-IC was the most sensitive of the diagnostic methods. It is possible that the IC findings in the 17 patients without a confirmatory test are false-positive reactions or that the lack of a confirmatory finding is only due to the poor diagnostic efficiency of the other methods. Several lines of evidence support the hypothesis that the presence of pneumolysin-IC is diagnostic for Pnc pneumonia. We could not detect pneumolysin antibodies in IC isolated from 120 sera of healthy persons even though some of these sera contained free pneumolysin antibodies in very high titers (around 1/lO,OOO). In addition, false-positive IC findings due to carriage of Pnc in the upper respiratory tract can possibly be excluded by the fact that the carriage rate for Pnc in Finnish children is around 20% (unpublished data) and the 98 control sera obtained from children were all negative for pneumolysinIC. These findings suggest that the presence of pneumolysin-IC is a strong indication of Pnc infection. Pneumolysin is an intracellular protein toxin of Pnc and it is liberated from Pnc cells after their lysis I2. We can thus speculate that in bacteremic infections or in lung infections, when bacterial products have an easy access to circulation, IC containing pneumolysin and its antibodies are easily formed. When Pnc was indicated by any of the diagnostic methods pneumolysin-IC were present in 57% of the acute sera and in 83% of the paired sera taken 5-8 days apart. These data suggest that the detection of IC in acute serum samples could be used as a rapid diagnostic method. Although sputum studies might provide etiological diagnosis even more rapidly than the demonstration of pneumolysin-IC, by which the result can be obtained only on the following day, sputum specimens are not obtained from all patients and especially often not from those with the most severe infection. The handling and treatment of sputum samples is also laborious and time-consuming. The second advantage of IC determinations, especially when compared to normal antibody assays, is that paired sera taken not more than 1 week apart can be used for these studies and thus no extra hospital visit for convalescent serum is needed. In addition, it allows the specific antimicrobial therapy for most of the patients with Pnc pneumonia. In conclusion, the data presented strongly suggest that detection of pneumolysin antibodies in circulating immune complexes is a sensitive, reliable diagnostic method for pneumococcal pneumonia that can also be used as a rapid diagnostic test. The detection of specific antibodies from circulating immune complexes, earlier shown to be useful in Lyme disease6 and now in pneumococcal pneumonia, gives a new perspective for the etiological diagnosis of severe bacterial infections. Acknowledgements
We are grateful to Teija Jaakkola RN, Raili Kalliokokoski RN and Kaija Vilen RN for their skilful technical assistance. These studies have been partly supported by the Medical Council, Academy of Finland. References 1. Kahn M, Kanclerski K, Granstriim M, Mijllby R. Diagnosis of pneumococcal pneumonia by enzyme-linked immunosorbent assay of antibodies to pneumococcal hemolysin. J Ciin Microbial 1987; 25: 2269.
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2. Kanclerski K, Blomqvist S, Granstrom M, Mijllby R. Serumantibodiesto pneumolysinin patientswith pneumonia.J Clin Microbial 1988;26: 96-100. 3. Jalonen E, Paton JC, Koskela M, Kerttula Y, Leinonen M. Measurementof antibody responseto pneumolysin-a promisingmethod for the presumptiveetiological diagnosisof pneumococcalpneumonia.J Infect 1989;19: 127-34. 4. Ortqvist A, Grillner L, Hedlund J et al. Outcome and prognostic factors in communityacquiredpneumonia.Eur RespJ 1990;3: 1105-13. 5. LeinonenM. Detection of pneumococcalcapsularpolysaccharideantigensby latex agglutination, counterimmunoelectrophoresis and radioimmunoassayin middle ear exudatesin acute otitis media.J Clin Microbial 1980;11: 135-40. 6. Schutzer SE, Coyle PK, BelmanA, Golightly MG, Drulle J. Sequestrationof antibody to Borrelia burgdorferi in immunecomplexesin seronegativeLyme disease. Lancet 1990;i: 31215. 7. CohenRS. Useof predictive value theory in clinical immunology.In: RoseNR, FriedmanH, Fahey JL, eds. Manual of Clinical Laboratory Immunology. Washington D.C.: American Society for Microbiology, 1986:96670. 8. MacFarlane JT, Ward MJ, Finch RG, Macrae D. Hospital study of adult communityacquiredpneumonia.Lancet 1982;ii: 255-8. 9. Berntsson E, Blomberg J, Lagergard T, Trollfors B. Etiology of community-acquired pneumoniain patientsrequiring hospitalization, Eur J Clin Microbial 1985;4: 268-72. 10. HolmbergH. Etiology of community-acquiredpneumoniain hospitaltreated patients.Stand J Inf Dis 1987;19: 491-501. 1I. Marrie TJ, Durant H, Yates L. Community-acquiredpneumoniarequiring hospitalization. Rev Inf Dis 1989;11: 586-99. 12. Berry AM, Lock RA, HansmanD, Paton JC. Contribution of autolysin to virulence of Streptococcus pneumoniae. Infect Immunol 1989;57: 232430. (Manuscript accepted 3 May 1991)