JOURNAL OF CLINICAL MICROBIOLOGY, May 1992,
Vol. 30, No. 5
p. 1198-1204 0095-1137/92/051198-07$02.00/0 Copyright © 1992, American Society for Microbiology
Enzyme Immunoassay for Detection of Immunoglobulin M (IgM) and IgG Antibodies to Mycoplasma pneumoniae S0REN A. ULDUM,* J0RGEN S. JENSEN, JAN S0NDERGARD-ANDERSEN,t AND KLAUS LIND Mycoplasma Laboratory, Neisseria Department, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen S, Denmark Received 15 October 1991/Accepted 28 January 1992
An enzyme immunoassay (ETA) for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae was developed. The EIA was evaluated on the basis of results in the M. pneumoniae complement fixation (MPCF) test and the cold agglutinin test. Serum samples from 430 patients with respiratory infections of known or unknown etiology, from 91 healthy children and adults and from 20 patients with rheumatoid factor, were investigated. By the criteria chosen for positive diagnostic EIA values, we found that the combined measurement of specific IgM and IgG gave a specificity of 99.7% and a sensitivity of 97.8%. If only IgM antibodies were measured, the specificity was 100% and the sensitivity was 88%. For IgG alone the specificity was 99.7%, but the sensitivity was only 46% because of the high EIA cutoff value chosen for IgG. We found no false positives among serum samples from patients with non-M. pneumoniae respiratory infection of known etiology, and there were no false IgM positives due to rheumatoid factor. In some cases the IgM EIA results became positive earlier in the course of illness than the MPCF titer. While children and teenagers responded predominantly with IgM antibodies, patients older than 40 years often had an IgG response only (56% of cases), probably because of reinfection. We conclude that this ETA is a good alternative to the combined MPCF and cold agglutinin tests in the diagnosis of M. pneumoniae infection.
Mycoplasma pneumoniae is a common cause of lower respiratory tract disease, especially in children and young adults. The diagnosis is in most cases confirmed by serology. M. pneumoniae is difficult to culture because of its fastidious nature, and growth is too slow for diagnostic use. New rapid methods for detection of the organism by immunological techniques (8, 18), gene probe (8, 17), and polymerase chain reaction (13) are promising but need further evaluation for diagnostic use. The M. pneumoniae complement fixation (MPCF) test based on a glycolipid antigen has been the most commonly used serological test since it was described by Kenny and Grayston in 1965 (14). The test has been evaluated and was found to be both sensitive and specific (15). Although its specificity has been questioned in patients with pancreatitis (19) and other extrapulmonary manifestations (16, 27), showing titer rises to low or moderate antibody levels, these conditions do not generally motivate the clinician to have an MPCF test made. The cold agglutinin (CA) test has been retained for many years as a supplement to the MPCF test. CAs develop in more than half of the patients with M. pneumoniae infection (22), and being immunoglobulin M (IgM) antibodies, they are the first to be detected and the first to disappear again (6). However, CA may be produced by patients without M. pneumoniae infection (24). Several enzyme immunoassays (EIA) for the serological diagnosis of M. pneumoniae infection have been published in the last 10 years. They include assays either for the determination of specific IgG only (30), for both IgG and IgM (7, 11, 12, 25, 31), and assays for IgM only (,u-capture) (9, 34). However, most of them are not evaluated with respect
to predictive diagnostic values in a routine diagnostic setting. Some lack sufficient sensitivity and/or specificity, and interpretation of the results is often hampered by patients' preexisting M. pneumoniae antibodies (7, 11, 25, 30). In this report we present an evaluation of an EIA for the detection of specific IgM and IgG to M. pneumoniae based on a comparison with results from the MPCF test combined with the CA test. The predictive values of a diagnostic positive and a negative EIA in different epidemiological situations are discussed.
MATERIALS AND METHODS Antigen preparation for EIA. M. pneumoniae Mac was grown at 37°C in 800-ml Nunclon plastic flasks (Nunc, Roskilde, Denmark) in 150 ml of Hayflick's medium (23) with 0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid) buffer. When a dense layer of colonies appeared and the color was turning orange, the medium was removed. Plast-adherent mycoplasmas were washed three times with 0.9% (wt/vol) NaCl with penicillin G (500 U/ml) and harvested by shaking the flasks with sterile glass beads. The organisms were then washed twice by suspension in 0.9% NaCl with penicillin and centrifugation at 39,000 x g for 10 min at 6°C. The pellets were suspended in 0.5 ml of saline per flask with penicillin and stored at -80°C. Aliquots of 300 pul of the organisms were thawed, and 150 ,ul of 0.9% (wt/vol) NaCl and 50 ,ul of 10% (wt/vol) sodium dodecyl sulfate (SDS) (lot L-4509; Sigma Chemical Co., St. Louis, Mo.) were added per 300 ,ul, giving a final SDS concentration of 1% (wt/vol). The suspension was mixed well and incubated at room temperature for 30 min. The cell lysate was centrifuged at 30,000 x g at 15°C for 10 min. The supernatant, containing about 10 mg of protein per ml as estimated by the method of Lowry (24a), was stored at -80°C until use as antigen in the EIA. Conjugates. Horseradish peroxidase-conjugated rabbit
* Corresponding author. t Present address: Novo-Nordisk A/S, Immunkemisk Afdeling,
DK-2820 Gentofte, Denmark.
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EIA FOR DETECTION OF M. PNEUMONIAE ANTIBODIES
anti-human IgM (code P215) and anti-human IgG (code P214) were both from DAKO, Copenhagen, Denmark. EIA procedure. After optimization with regard to antigen dilution, serum dilution, enzyme-substrate reaction time, and type of microtiter plAte, the following procedure for determining IgM and IgG antibodies to M. pneumoniae was found to be the most appropriate. Each well except all outer wells of the microtiter plates (PolySorp; Nunc, Roskilde, Denmark) was coated with 100 RI of antigen diluted 1:1,000 in 0.05 M carbonate buffer (pH 9.6) and incubated at 4°C overnight or longer, covered with parafilm (American National Can, Greenwich, Conn.). Under these conditions the plates could be stored for at least 2 weeks. The plates were washed twice with phosphate-buffered saline (PBS; pH 7.4) with 0.05% (vol/vol) Tween 20 (article 822184; E. Merck AG, Darmstadt, Germany) (PBS-T), and then postcoated with 120 ,ul of a 5% (wt/vol) solution of skim bovine milk powder (Irma A/S, R0dovre, Denmark) in PBS-T (PBS-T-M) at room temperature for 30 min. The wells were washed as described above, and 90 [lI of PBS-T-M was added to each well. Ten microliters of the serum to be tested, diluted 1:50 in PBST-M, was then added to each of two wells in two plates, giving a final serum dilution of 1:500. The plates were covered with parafilm and incubated for 1.5 h at 37°C on a shaking table and washed four times in PBS-T, and then 100 ptl of the two conjugates diluted 1:2,000 in PBS-T-M was added to the relevant plates. The plates were incubated for 1 h as described above and washed three times in PBS-T and once in deionized water. The wells were then filled with citrate buffer (pH 5), incubated for 2 min, and emptied, and 100 ,ul of substrate solution (0.38 mg of 1,2-phenylenediamine-dihydrochloride [78440; Fluka AG, Buchs, Switzerland], 50 ml of citrate buffer, pH 5, and 20 pul of 30% H202) was added to each well. After incubation for 6 min, the reaction was stopped by adding 150 ,ul of 1 N H2SO4 to each well. The A490 was read with a photometer (ImmunoReader NJ 2000; InterMed, Tokyo, Japan). The blank was a well coated with antigen and incubated with the conjugates only. Reference sera and calculation of EIA values. Five reference serum samples were included in duplicate in each experiment. Each of the five serum samples were given both an IgM and an IgG EIA reference value, calculated as the mean optical density (OD) value of 10 experiments multiplied by 100. The EIA reference (ref.) values were as follows: ref. 1, IgM, 1.8, and IgG, 1.7; ref. 2, IgM, 27, and IgG, 24; ref. 3, IgM, 63, and IgG, 71; ref. 4, IgM, 93, and IgG, 106; ref. 5, IgM, 144, and IgG, 146. The OD values for each of the five reference serum samples (mean of two wells) were used to construct a calibration curve (OD values versus their respective EIA reference values). The line of best fit was used to determine the EIA values of each serum sample tested (mean of two wells). The linear regression and the test results (EIA values) were calculated by a computer program made in SuperCalc 5.0 (Computer Associates International, Inc.). Sera with values above 180 were given the value >180 because OD values above 1.8 were not linear. For validation of the experiment the correlation coefficient (r2) of the line should be >0.95, and the calculated values for ref. 1 should be 120. Reproducibility. Interassay reproducibility of the EIA was estimated by calculating the mean EIA value, the standard deviation, and the coefficient of variation for 24 different test runs for the five reference serum samples, four control serum samples from patients with MPCF titers of .64, and two control pools of sera, namely, pool E (sera from children
1199
c 5 years old with MPCF titers of 64 between two consecutively collected serum samples or if single or standing titers were >512; single or standing titers from 64 to 256 were considered presumptive of a current or past infection. CA test. CA titration was performed as previously described (20). In brief, sera were diluted by twofold dilutions in 0.4 ml of VBS (pH 7.6) from 1:16 to 1:2,048. A 2.5% suspension (25 pul) of washed human group OI erythrocytes was added to each tube. The tubes were shaken and then incubated overnight at 4°C. After a light shake, the tubes were observed against an illuminated frosted green glass plate, and the titer was read as the reciprocal of the highest serum dilution giving visible agglutination. A titer of 264 was considered positive. Although nonspecific, a positive CA titer together with a positive MPCF titer supports the diagnosis of a current M. pneumoniae infection. Cultivation of M. pneumoniae from patients. Cotton-tip swabs were taken from the naso-oropharynx and immediately immersed into a vial with 3 ml of mycoplasma broth (SP4 medium) (29). The vial was transported within 24 h to the laboratory, where two agar plates were streaked, and two vials with broth were inoculated with 0.1 ml each; the two media were the classical Hayflick's medium and SP4 medium. After incubation of the agar plates at 37°C in a moist atmosphere of 5% CO2 and 8% 02, growth of M. pneumoniae was identified by the disk growth inhibition test
(5).
Groups of human serum samples. Sera from a total of 541 patients and healthy persons were included. The patients were separated into two categories as follows: one of patients with respiratory tract infections and positive MPCF test results, divided into three groups, and the other of patients and healthy persons with MPCF negative sera, divided into four groups. The categories and groups are described below. MPCF-positive serum samples. (i) Group 1. At least two consecutive serum samples from each of 95 patients with respiratory infections (mostly pneumonia), all showing a
1200
J. CLIN. MICROBIOL.
ULDUM ET AL.
TABLE 1. Distribution of persons in the four MPCF-negative groups with regard to IgM EIA values value
0-9 10-19 20-29 Total
No. (%) in following group:
No. (%) in following group:
IgM EIA 4
(n
=
160)
Sa (n
=
42)
6
(n
=
20)
7
(n
=
TABLE 2. Distribution of persons in the four MPCF-negative groups with regard to IgG EIA values
IgG EIA
91)
T=t313)
140 (88) 20 (12) 0 (0)
36 (86) 6 (14) 0 (0)
19 (95) 1 (5) 0 (0)
77 (85) 14 (15) 0 (0)
272 (87) 41 (13) 0 (0)
160 (100)
42 (100)
20 (100)
91 (100)
313 (100)
a Values for convalescent-phase serum only.
>fourfold titer rise to at least 64 in the MPCF test; median age, 21 years (range, 3 to 78 years). (ii) Group 2. Single serum samples from 84 patients with MPCF titers of .512; median age, 22 years (range, 1 to 62 years). (iii) Group 3. Single serum samples from 49 patients with MPCF titers from 64 to 256; median age, 28 years (range, 1 to 73 years). MPCF-negative serum samples. (i) Group 4. Single serum samples from 160 patients with respiratory tract infection of unknown etiology with MPCF titers of 60 was considered presumptive of a current or past infection. Specificity. The EIA results for MPCF-negative sera (groups 4 to 7) are presented as number and percentage of persons with IgM values in Table 1 and IgG values in Table 2, stratified in various EIA ranges. None of the sera from the patients with non-M. pneumoniae respiratory infections in
value
0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-100 >100
Total
6(n20) 4 (n =160) 5 5a(n42) n=4)6n=207(n=9 =313) 7(n=91)(nTotal
82 51 17 4 4 1 1
(51)
(32) (11) (2.5) (2.5) (0.5) (0.5)
26 (62) 15 (36) 1 (2)
14 (70) 5 (25)
63 21 6 1
(69) (23) (7) (1)
1 (5)
160 (100)
42 (100)
20 (100)
91 (100)
185 92 24 5 4 1 1 0 1
(59) (29) (8) (2) (1) (0.3) (0.3) (0) (0.3)
313 (100)
a Values for convalescent-phase serum only.
group 5 showed a significant increase in EIA value, indicating absence of cross-reactivity between the causative microorganisms and M. pneumoniae protein antigen. Table 1 shows that sera from all the MPCF-negative groups had IgM EIA values of 8 [10]) and a MPCF titer of 32 had a diagnostic IgG EIA value of 103 (IgM EIA = 19). With one false diagnostic positive serum the specificity for measuring IgG was 99.7% (312/313 x 100). For patients with non-M. pneumoniae infection and healthy adults and children (groups 5 and 7), the specificity was 100% with no IgG values of >40. Sensitivity. EIA results for consecutive sera from patients with respiratory infections and diagnostic MPCF titer rises (group 1) are shown in Table 3, and single serum samples with MPCF titers of .512 (group 2) are shown in Table 4. The results are grouped according to number and percentage of patients with positive EIA values and/or increases in values and are stratified according to positive and negative CA titers. It can be seen from Tables 3 and 4 that there is an association between CA-positive and IgM EIA-positive reactions and between CA-negative and IgG-positive reactions, respectively. If groups 1 and 2 are calculated together, 175 of 179 patient sera had diagnostic positive EIA values, giving a sensitivity of 97.8%. Among these, 18 (10%) were exclusively IgG positive, 93 (53%) were exclusively IgM positive, and 64 (37%) were both IgG and IgM positive. Of the patients, 156 (87%) had a diagnostic value by a single criterion: an IgM EIA value of .60. Among consecutive sera in group 1, 63 (68%) of 92 EIA diagnostic positive patients developed significant increases in IgM and/or IgG values, and 4 paired sera were positive only by this criterion. Of another 4 patients in groups 1 and 2, there were three EIA presumptive positives and one negative. The latter was a 12-year-old boy with pneumonia whose first serum had MPCF and CA titers of < 16 and < 16, respectively, and IgM and IgG EIA values of 6 and 15, respectively, and whose second serum had MPCF and CA titers of 256 and 64
34 (69)
.32
15 (31)
IgM and IgG
IgG
IgM
30-59
. 60
60-99
2 (6) 0 (0)
25 (74)
1 (3) 2 (13)
4 (27)
.100
Total
Diag"
2 (6) 4 (27)
30 (88) 6 (67)
27 (79) 6 (53)
6 (12) 40 (82) 35 (71) Total 49 (100) 2 (4) 29 (59) 3 (6) a See footnote a of Table 3 for descriptions of criteria. b Diag, number of patients diagnostic positive for an IgM value of .60 and/or an IgG value of .100.
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ULDUM ET AL.
J. CILIN. MIC ROBIOI. TABLE 7. Interassay reproducibility for the EIA"
TABLE 6. MPCF-positive patients in groups 1, 2, and 3 with low and high EIA values and with positive CA titers
Age (yr)
IgM
patients
EIA value
30-59
0-9 10-19 20-29 30-39 40-49 >50
Total
50 51
47 50 16 14 228
8 2 2 2 0 0
3 (7)
.60
88 90 91 78 44 36
81(185)
IgG
EIA value
.60)
30-59
18 25 19 16 6 21
20 (45)
16 25 51 58 63 71
41 (94)
Mean'
CV'
SD
Serum
%' (no.) of patients with following result titer
of .64
92 86
91 84 56 64
85 (193)
ref. 1 ref. 2 ref. 3 ref. 4 ref. 5 Pool E Pool A 8150 6948 17648 17981
only produced IgG antibodies, probably as a result of reinfection; of 30 MPCF-positive patients older than 39 years of age, 56% had negative IgM EIA values of below 30 (Table 6), and about 12% of all MPCF- and EIA-positive patients had IgG responses only. Other good reasons for measuring both IgM and IgG M. pneurnoniae-specific antibodies are (i) The level of IgG antibodies normally rises later than the IgM level, especially in children (2, 11, 26, 31), and (ii) some patients continue to produce IgM predominantly during illness (2, 25). We found that 66% of MPCF-positive children younger than 10 years had negative IgG EIA values of 150) reduced the IgM reaction significantly (by 20 to 40%), thereby causing a risk of false-negative IgM reactions. However, the EIA result summary would still be positive because of the high IgG value (data not given). Rabbit antisera raised against Mycoplasma genitalium (23) showed reactions with the M. pneurnoniae antigen used in this EIA when an anti-rabbit immunoglobulin conjugate was employed, and MPCF-positive human sera showed reactions with M. genitalium antigen preparcd in the same way as the M. pneumoniae antigen (unpublished observations). Obviously, the EIA would not discriminate between human antibodies to the two cross-reacting species (23). Although M. genitaliumn has bcen isolated from throat swabs of patients (1), it is not yet known whether the organism is a human pathogen. In the search for possible M. genitalium infections, we performed immunoblotting with several EIApositive sera against both M. ptnewunoniae and M. genitaliiwn; however, all positivc sera showed reactions with both antigens; a few stronger reactions seen with M. genitalium were inconclusive. We have not considered the possibility of M. genitaliumn infection in the evaluation of the specificity of the EIA. We believe that this EIA, which has a specificity of 99.7%, a sensitivity of 97.8&% compared with the MPCF test, and a relative good reproducibility, is a realistic diagnostic alternative to the MPCF test evcl if it is supplemented with the CA test. REFERENCES 1. Baseman, J. B., S. F. Dallo, J. G. Tully, and D. L. Rose. 1988. Isolation and characterization of Mycoplasma genitaliumn strains from the human respiratory tract. J. Clin. Microbiol.
26:2266-2269. 2. Biberfeld, G. 1971. Antibody responses in Mycoplasma pneumoniae infection in relation to serum immunoglobulins, especially IgM. Acta Pathol. Microbiol. Scand. Sect. B 79:620-634. 3. Chamberlain, P., and A. A. Saeed. 1983. A study of the specific IgM antibody response in Mycoplasima pneumoniae infection in man. J. Hyg. Camb. 90:207-211. 4. Champsaur, H., M. Fattal-German, and R. Arranhado. 1988. Sensitivity and specificity of viral immunoglobulin M determination by indirect enzyme-linked immunosorbent assay. J. Clin. Microbiol. 26:328-332. 5. Clyde, W. A., Jr. 1964. Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immunol. 92:958965. 6. Denny, F. W., W. A. Clyde, Jr., and W. P. Glezen. 1971. Mycoplasma pneumoniae disease: clinical spectrum, pathophysiology, epidemiology, anid control. J. Infect. Dis. 123:7492. 7. Dussaix, E., A. Slim, and P. Tournier. 1983. Comparison of enzyme-linked immunosorbent assay and complement fixation test for detection of MycoIplasma pneumoniae antibodies. J. Clin. Pathol. 36:228-232. 8. Harris R., B. P. Marmion, G. Varkanis, T. Kok, B. Lunn, and J. Martin. 1988. Laboratory diagnosis of Mycoplasma pneumoniae infection. 2. Comparison of methods for the direct detection of specific antigen or nucleic acid sequences in respiratory exudates. Epidemiol. Infect. 101:685--694.
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9. Hirschberg, L., A. Krook, C. A. Pettersson, and T. Vikerfors. 1988. Enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae specific immunoglobulin M. Eur. J. Clin. Microbiol. Infect. Dis. 7:420-423. 10. H0ier-Madsen, M., L. P. Nielsen, and S. M0ller. 1986. Bestemmelse af IgM-Rheumafaktorer ved Enzyme-linked immunosorbent Assay (ELISA). Ugeskr. Laeg. 148:2018-2021. 11. Jacobs, E., A. Bennewitz, and W. Bredt. 1986. Reaction pattern of human anti-Mycoplasma pneumoniae antibodies in enzymelinked immunosorbent assays and immunoblotting. J. Clin. Microbiol. 23:517-522. 12. Jacobs, E., K. Fuchte, and W. Bredt. 1986. A 168-kilodalton protein of Mycoplasma pneumoniae used as antigen in a dot enzyme-linked immunosorbent assay. Eur. J. Clin. Microbiol. Infect. Dis. 5:435-440. 13. Jensen, J. S., J. S0ndergard-Andersen, S. A. Uldum, and K. Lind. 1989. Detection of Mycoplasma pneumoniae in simulated clinical samples by polymerase chain reaction. APMIS 97:10461048. 14. Kenny, G. E., and J. T. Grayston. 1965. Eaton pleuropneumoniae-like organism (Mycoplasma pneumoniae) complement-fixing antigen: extraction with organic solvents. J. Immunol. 95:19-25. 15. Kenny, G. E., G. G. Kaiser, M. K. Cooney, and H. M. Foy. 1990. Diagnosis of Mycoplasma pneumoniae pneumonia: sensitivities and specificities of serology with lipid antigen and isolation of the organism on soy peptone medium for identification of infections. J. Clin. Microbiol. 28:2087-2093. 16. Kleemola, M., and H. Kfyhty. 1982. Increase in titers of antibodies in patients with purulent meningitis. J. Infect. Dis. 146:284-288. 17. Kleemola, S. R. M., J. E. Karjalainen, and R. K. H. Raty. 1990. Rapid diagnosis of Mycoplasma pneumoniae infection: clinical evaluation of a commercial probe test. J. Infect. Dis. 126:70-75. 18. Kok, T. W., G. Varkanis, B. P. Marmion, J. Martin, and A. Esterman. 1988. Laboratory diagnosis of Mycoplasma pneumoniae infection. 1. Direct detection of antigen in respiratory exudates by enzyme immunoassay. Epidemiol. Infect. 101:669684. 19. Leinikki, P. O., P. Panzar, and H. Tykka. 1978. Immunoglobulin M response against Mycoplasma pneuinoniae lipid antigen in patients with acute pancreatitis. J. Clin. Microbiol. 8:113-118. 20. Lind, K., and M. W. Bentzon. 1976. Epidemics of Mycoplasma pneumoniae infection in Denmark from 1958 to 1974. Int. J. Epidemiol. 5:267-277. 21. Lind, K., and M. W. Bentzon. 1988. Changes in the epidemiological pattern of Mycoplasma pneumoniae infection in Denmark. A 30 years survey. Epidemiol. Infect. 101:377-386. 22. Lind, K., and M. W. Bentzon. 1991. Ten and a half years seroepidemiology of Mycoplasma pneumoniae infection in Denmark. Epidemiol. Infect. 107:189-199. 23. Lind, K., B. 0. Lindhardt, H. J. Schutten, J. Blom, and C. Christiansen. 1984. Serological cross-reactions between Mycoplasma genitalium and Mycoplasma pnelumoniae. J. Clin. Microbiol. 20:1036-1043. 24. Lind, K., T. J. Ravn, and J. M0ller. 1970. Occurrence of Mycoplasma pneumoniae infection in patients hospitalized with acute respiratory illness. Acta Pathol. Microbiol. Scand. Sect. B 78:6-14. 24a.Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 25. Mitzutani, H., and H. Mitzutani. 1983. Immunologic responses in patients with Mycoplasma pneumoniae infections. Am. Rev. Respir. Dis. 127:175-179. 26. Moule, J. H., E. 0. Caul, and T. G. Wreghitt. 1987. The specific IgM response to Mycoplasma pneumoniae infection: interpretation and application to early diagnosis. Epidemiol. Infect. 99:685-692. 27. Ponkii, A., T. Ponka, S. Sarna, and K. Penttinen. 1981. Questionable specificity of lipid antigen in the Mycoplasma pneumoniae complement fixation test in patients with extrapulmonary manifestations. J. Infect. 3:332-338.
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28. Sillis, M. 1990. The limitations of IgM assays in the serological diagnosis of Mycoplasma pneumoniae infections. J. Med. Microbiol. 33:253-258. 29. Tully, J. G., R. F. Whitcomb, H. F. Clark, and D. L. Williamson. 1977. Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma. Science 195:892-894. 30. Uldum, S. A., J. S0ndergArd-Andersen, J. S. Jensen, and K. Lind. 1990. Evaluation of a commercial enzyme immunoassay for detection of Mycoplasma pneumoniae specific immunoglobulin G antibodies. Eur. J. Clin. Microbiol. Infect. Dis. 9:221223. 31. van Griethuysen, A. J. A., R. de Graaf, J. A. M. van Druten, F. W. A. Heessen, J. T. M. van der Logt, and A. M. van Loon.
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1984. Use of the enzyme-linked immunosorbent assay for the early diagnosis of Mycoplasma pneumoniae infection. Eur. J. Clin. Microbiol. 3:116-121. 32. Vecchio, T. J. 1966. Predictive value of a single diagnostic test in unselected populations. N. Engl. J. Med. 274:1171-1173. 33. Vejtorp, M. 1980. The interference of IgM rheumatoid factor in enzyme-linked immunosorbent assays of rubella 1gM and IgG antibodies. J. Virol. Methods 1:1-9. 34. Wreghitt, T. G., and M. Sillis. 1985. A >.-capture ELISA for detecting Mycoplasma pneumoniae IgM: comparison with indirect immunofluorescence and indirect ELISA. J. Hyg. Camb. 94:217-227.
Vol. 30, No. 5
p. 1198-1204 0095-1137/92/051198-07$02.00/0 Copyright © 1992, American Society for Microbiology
Enzyme Immunoassay for Detection of Immunoglobulin M (IgM) and IgG Antibodies to Mycoplasma pneumoniae S0REN A. ULDUM,* J0RGEN S. JENSEN, JAN S0NDERGARD-ANDERSEN,t AND KLAUS LIND Mycoplasma Laboratory, Neisseria Department, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen S, Denmark Received 15 October 1991/Accepted 28 January 1992
An enzyme immunoassay (ETA) for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae was developed. The EIA was evaluated on the basis of results in the M. pneumoniae complement fixation (MPCF) test and the cold agglutinin test. Serum samples from 430 patients with respiratory infections of known or unknown etiology, from 91 healthy children and adults and from 20 patients with rheumatoid factor, were investigated. By the criteria chosen for positive diagnostic EIA values, we found that the combined measurement of specific IgM and IgG gave a specificity of 99.7% and a sensitivity of 97.8%. If only IgM antibodies were measured, the specificity was 100% and the sensitivity was 88%. For IgG alone the specificity was 99.7%, but the sensitivity was only 46% because of the high EIA cutoff value chosen for IgG. We found no false positives among serum samples from patients with non-M. pneumoniae respiratory infection of known etiology, and there were no false IgM positives due to rheumatoid factor. In some cases the IgM EIA results became positive earlier in the course of illness than the MPCF titer. While children and teenagers responded predominantly with IgM antibodies, patients older than 40 years often had an IgG response only (56% of cases), probably because of reinfection. We conclude that this ETA is a good alternative to the combined MPCF and cold agglutinin tests in the diagnosis of M. pneumoniae infection.
Mycoplasma pneumoniae is a common cause of lower respiratory tract disease, especially in children and young adults. The diagnosis is in most cases confirmed by serology. M. pneumoniae is difficult to culture because of its fastidious nature, and growth is too slow for diagnostic use. New rapid methods for detection of the organism by immunological techniques (8, 18), gene probe (8, 17), and polymerase chain reaction (13) are promising but need further evaluation for diagnostic use. The M. pneumoniae complement fixation (MPCF) test based on a glycolipid antigen has been the most commonly used serological test since it was described by Kenny and Grayston in 1965 (14). The test has been evaluated and was found to be both sensitive and specific (15). Although its specificity has been questioned in patients with pancreatitis (19) and other extrapulmonary manifestations (16, 27), showing titer rises to low or moderate antibody levels, these conditions do not generally motivate the clinician to have an MPCF test made. The cold agglutinin (CA) test has been retained for many years as a supplement to the MPCF test. CAs develop in more than half of the patients with M. pneumoniae infection (22), and being immunoglobulin M (IgM) antibodies, they are the first to be detected and the first to disappear again (6). However, CA may be produced by patients without M. pneumoniae infection (24). Several enzyme immunoassays (EIA) for the serological diagnosis of M. pneumoniae infection have been published in the last 10 years. They include assays either for the determination of specific IgG only (30), for both IgG and IgM (7, 11, 12, 25, 31), and assays for IgM only (,u-capture) (9, 34). However, most of them are not evaluated with respect
to predictive diagnostic values in a routine diagnostic setting. Some lack sufficient sensitivity and/or specificity, and interpretation of the results is often hampered by patients' preexisting M. pneumoniae antibodies (7, 11, 25, 30). In this report we present an evaluation of an EIA for the detection of specific IgM and IgG to M. pneumoniae based on a comparison with results from the MPCF test combined with the CA test. The predictive values of a diagnostic positive and a negative EIA in different epidemiological situations are discussed.
MATERIALS AND METHODS Antigen preparation for EIA. M. pneumoniae Mac was grown at 37°C in 800-ml Nunclon plastic flasks (Nunc, Roskilde, Denmark) in 150 ml of Hayflick's medium (23) with 0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid) buffer. When a dense layer of colonies appeared and the color was turning orange, the medium was removed. Plast-adherent mycoplasmas were washed three times with 0.9% (wt/vol) NaCl with penicillin G (500 U/ml) and harvested by shaking the flasks with sterile glass beads. The organisms were then washed twice by suspension in 0.9% NaCl with penicillin and centrifugation at 39,000 x g for 10 min at 6°C. The pellets were suspended in 0.5 ml of saline per flask with penicillin and stored at -80°C. Aliquots of 300 pul of the organisms were thawed, and 150 ,ul of 0.9% (wt/vol) NaCl and 50 ,ul of 10% (wt/vol) sodium dodecyl sulfate (SDS) (lot L-4509; Sigma Chemical Co., St. Louis, Mo.) were added per 300 ,ul, giving a final SDS concentration of 1% (wt/vol). The suspension was mixed well and incubated at room temperature for 30 min. The cell lysate was centrifuged at 30,000 x g at 15°C for 10 min. The supernatant, containing about 10 mg of protein per ml as estimated by the method of Lowry (24a), was stored at -80°C until use as antigen in the EIA. Conjugates. Horseradish peroxidase-conjugated rabbit
* Corresponding author. t Present address: Novo-Nordisk A/S, Immunkemisk Afdeling,
DK-2820 Gentofte, Denmark.
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EIA FOR DETECTION OF M. PNEUMONIAE ANTIBODIES
anti-human IgM (code P215) and anti-human IgG (code P214) were both from DAKO, Copenhagen, Denmark. EIA procedure. After optimization with regard to antigen dilution, serum dilution, enzyme-substrate reaction time, and type of microtiter plAte, the following procedure for determining IgM and IgG antibodies to M. pneumoniae was found to be the most appropriate. Each well except all outer wells of the microtiter plates (PolySorp; Nunc, Roskilde, Denmark) was coated with 100 RI of antigen diluted 1:1,000 in 0.05 M carbonate buffer (pH 9.6) and incubated at 4°C overnight or longer, covered with parafilm (American National Can, Greenwich, Conn.). Under these conditions the plates could be stored for at least 2 weeks. The plates were washed twice with phosphate-buffered saline (PBS; pH 7.4) with 0.05% (vol/vol) Tween 20 (article 822184; E. Merck AG, Darmstadt, Germany) (PBS-T), and then postcoated with 120 ,ul of a 5% (wt/vol) solution of skim bovine milk powder (Irma A/S, R0dovre, Denmark) in PBS-T (PBS-T-M) at room temperature for 30 min. The wells were washed as described above, and 90 [lI of PBS-T-M was added to each well. Ten microliters of the serum to be tested, diluted 1:50 in PBST-M, was then added to each of two wells in two plates, giving a final serum dilution of 1:500. The plates were covered with parafilm and incubated for 1.5 h at 37°C on a shaking table and washed four times in PBS-T, and then 100 ptl of the two conjugates diluted 1:2,000 in PBS-T-M was added to the relevant plates. The plates were incubated for 1 h as described above and washed three times in PBS-T and once in deionized water. The wells were then filled with citrate buffer (pH 5), incubated for 2 min, and emptied, and 100 ,ul of substrate solution (0.38 mg of 1,2-phenylenediamine-dihydrochloride [78440; Fluka AG, Buchs, Switzerland], 50 ml of citrate buffer, pH 5, and 20 pul of 30% H202) was added to each well. After incubation for 6 min, the reaction was stopped by adding 150 ,ul of 1 N H2SO4 to each well. The A490 was read with a photometer (ImmunoReader NJ 2000; InterMed, Tokyo, Japan). The blank was a well coated with antigen and incubated with the conjugates only. Reference sera and calculation of EIA values. Five reference serum samples were included in duplicate in each experiment. Each of the five serum samples were given both an IgM and an IgG EIA reference value, calculated as the mean optical density (OD) value of 10 experiments multiplied by 100. The EIA reference (ref.) values were as follows: ref. 1, IgM, 1.8, and IgG, 1.7; ref. 2, IgM, 27, and IgG, 24; ref. 3, IgM, 63, and IgG, 71; ref. 4, IgM, 93, and IgG, 106; ref. 5, IgM, 144, and IgG, 146. The OD values for each of the five reference serum samples (mean of two wells) were used to construct a calibration curve (OD values versus their respective EIA reference values). The line of best fit was used to determine the EIA values of each serum sample tested (mean of two wells). The linear regression and the test results (EIA values) were calculated by a computer program made in SuperCalc 5.0 (Computer Associates International, Inc.). Sera with values above 180 were given the value >180 because OD values above 1.8 were not linear. For validation of the experiment the correlation coefficient (r2) of the line should be >0.95, and the calculated values for ref. 1 should be 120. Reproducibility. Interassay reproducibility of the EIA was estimated by calculating the mean EIA value, the standard deviation, and the coefficient of variation for 24 different test runs for the five reference serum samples, four control serum samples from patients with MPCF titers of .64, and two control pools of sera, namely, pool E (sera from children
1199
c 5 years old with MPCF titers of 64 between two consecutively collected serum samples or if single or standing titers were >512; single or standing titers from 64 to 256 were considered presumptive of a current or past infection. CA test. CA titration was performed as previously described (20). In brief, sera were diluted by twofold dilutions in 0.4 ml of VBS (pH 7.6) from 1:16 to 1:2,048. A 2.5% suspension (25 pul) of washed human group OI erythrocytes was added to each tube. The tubes were shaken and then incubated overnight at 4°C. After a light shake, the tubes were observed against an illuminated frosted green glass plate, and the titer was read as the reciprocal of the highest serum dilution giving visible agglutination. A titer of 264 was considered positive. Although nonspecific, a positive CA titer together with a positive MPCF titer supports the diagnosis of a current M. pneumoniae infection. Cultivation of M. pneumoniae from patients. Cotton-tip swabs were taken from the naso-oropharynx and immediately immersed into a vial with 3 ml of mycoplasma broth (SP4 medium) (29). The vial was transported within 24 h to the laboratory, where two agar plates were streaked, and two vials with broth were inoculated with 0.1 ml each; the two media were the classical Hayflick's medium and SP4 medium. After incubation of the agar plates at 37°C in a moist atmosphere of 5% CO2 and 8% 02, growth of M. pneumoniae was identified by the disk growth inhibition test
(5).
Groups of human serum samples. Sera from a total of 541 patients and healthy persons were included. The patients were separated into two categories as follows: one of patients with respiratory tract infections and positive MPCF test results, divided into three groups, and the other of patients and healthy persons with MPCF negative sera, divided into four groups. The categories and groups are described below. MPCF-positive serum samples. (i) Group 1. At least two consecutive serum samples from each of 95 patients with respiratory infections (mostly pneumonia), all showing a
1200
J. CLIN. MICROBIOL.
ULDUM ET AL.
TABLE 1. Distribution of persons in the four MPCF-negative groups with regard to IgM EIA values value
0-9 10-19 20-29 Total
No. (%) in following group:
No. (%) in following group:
IgM EIA 4
(n
=
160)
Sa (n
=
42)
6
(n
=
20)
7
(n
=
TABLE 2. Distribution of persons in the four MPCF-negative groups with regard to IgG EIA values
IgG EIA
91)
T=t313)
140 (88) 20 (12) 0 (0)
36 (86) 6 (14) 0 (0)
19 (95) 1 (5) 0 (0)
77 (85) 14 (15) 0 (0)
272 (87) 41 (13) 0 (0)
160 (100)
42 (100)
20 (100)
91 (100)
313 (100)
a Values for convalescent-phase serum only.
>fourfold titer rise to at least 64 in the MPCF test; median age, 21 years (range, 3 to 78 years). (ii) Group 2. Single serum samples from 84 patients with MPCF titers of .512; median age, 22 years (range, 1 to 62 years). (iii) Group 3. Single serum samples from 49 patients with MPCF titers from 64 to 256; median age, 28 years (range, 1 to 73 years). MPCF-negative serum samples. (i) Group 4. Single serum samples from 160 patients with respiratory tract infection of unknown etiology with MPCF titers of 60 was considered presumptive of a current or past infection. Specificity. The EIA results for MPCF-negative sera (groups 4 to 7) are presented as number and percentage of persons with IgM values in Table 1 and IgG values in Table 2, stratified in various EIA ranges. None of the sera from the patients with non-M. pneumoniae respiratory infections in
value
0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-100 >100
Total
6(n20) 4 (n =160) 5 5a(n42) n=4)6n=207(n=9 =313) 7(n=91)(nTotal
82 51 17 4 4 1 1
(51)
(32) (11) (2.5) (2.5) (0.5) (0.5)
26 (62) 15 (36) 1 (2)
14 (70) 5 (25)
63 21 6 1
(69) (23) (7) (1)
1 (5)
160 (100)
42 (100)
20 (100)
91 (100)
185 92 24 5 4 1 1 0 1
(59) (29) (8) (2) (1) (0.3) (0.3) (0) (0.3)
313 (100)
a Values for convalescent-phase serum only.
group 5 showed a significant increase in EIA value, indicating absence of cross-reactivity between the causative microorganisms and M. pneumoniae protein antigen. Table 1 shows that sera from all the MPCF-negative groups had IgM EIA values of 8 [10]) and a MPCF titer of 32 had a diagnostic IgG EIA value of 103 (IgM EIA = 19). With one false diagnostic positive serum the specificity for measuring IgG was 99.7% (312/313 x 100). For patients with non-M. pneumoniae infection and healthy adults and children (groups 5 and 7), the specificity was 100% with no IgG values of >40. Sensitivity. EIA results for consecutive sera from patients with respiratory infections and diagnostic MPCF titer rises (group 1) are shown in Table 3, and single serum samples with MPCF titers of .512 (group 2) are shown in Table 4. The results are grouped according to number and percentage of patients with positive EIA values and/or increases in values and are stratified according to positive and negative CA titers. It can be seen from Tables 3 and 4 that there is an association between CA-positive and IgM EIA-positive reactions and between CA-negative and IgG-positive reactions, respectively. If groups 1 and 2 are calculated together, 175 of 179 patient sera had diagnostic positive EIA values, giving a sensitivity of 97.8%. Among these, 18 (10%) were exclusively IgG positive, 93 (53%) were exclusively IgM positive, and 64 (37%) were both IgG and IgM positive. Of the patients, 156 (87%) had a diagnostic value by a single criterion: an IgM EIA value of .60. Among consecutive sera in group 1, 63 (68%) of 92 EIA diagnostic positive patients developed significant increases in IgM and/or IgG values, and 4 paired sera were positive only by this criterion. Of another 4 patients in groups 1 and 2, there were three EIA presumptive positives and one negative. The latter was a 12-year-old boy with pneumonia whose first serum had MPCF and CA titers of < 16 and < 16, respectively, and IgM and IgG EIA values of 6 and 15, respectively, and whose second serum had MPCF and CA titers of 256 and 64
34 (69)
.32
15 (31)
IgM and IgG
IgG
IgM
30-59
. 60
60-99
2 (6) 0 (0)
25 (74)
1 (3) 2 (13)
4 (27)
.100
Total
Diag"
2 (6) 4 (27)
30 (88) 6 (67)
27 (79) 6 (53)
6 (12) 40 (82) 35 (71) Total 49 (100) 2 (4) 29 (59) 3 (6) a See footnote a of Table 3 for descriptions of criteria. b Diag, number of patients diagnostic positive for an IgM value of .60 and/or an IgG value of .100.
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ULDUM ET AL.
J. CILIN. MIC ROBIOI. TABLE 7. Interassay reproducibility for the EIA"
TABLE 6. MPCF-positive patients in groups 1, 2, and 3 with low and high EIA values and with positive CA titers
Age (yr)
IgM
patients
EIA value
30-59
0-9 10-19 20-29 30-39 40-49 >50
Total
50 51
47 50 16 14 228
8 2 2 2 0 0
3 (7)
.60
88 90 91 78 44 36
81(185)
IgG
EIA value
.60)
30-59
18 25 19 16 6 21
20 (45)
16 25 51 58 63 71
41 (94)
Mean'
CV'
SD
Serum
%' (no.) of patients with following result titer
of .64
92 86
91 84 56 64
85 (193)
ref. 1 ref. 2 ref. 3 ref. 4 ref. 5 Pool E Pool A 8150 6948 17648 17981
only produced IgG antibodies, probably as a result of reinfection; of 30 MPCF-positive patients older than 39 years of age, 56% had negative IgM EIA values of below 30 (Table 6), and about 12% of all MPCF- and EIA-positive patients had IgG responses only. Other good reasons for measuring both IgM and IgG M. pneurnoniae-specific antibodies are (i) The level of IgG antibodies normally rises later than the IgM level, especially in children (2, 11, 26, 31), and (ii) some patients continue to produce IgM predominantly during illness (2, 25). We found that 66% of MPCF-positive children younger than 10 years had negative IgG EIA values of 150) reduced the IgM reaction significantly (by 20 to 40%), thereby causing a risk of false-negative IgM reactions. However, the EIA result summary would still be positive because of the high IgG value (data not given). Rabbit antisera raised against Mycoplasma genitalium (23) showed reactions with the M. pneurnoniae antigen used in this EIA when an anti-rabbit immunoglobulin conjugate was employed, and MPCF-positive human sera showed reactions with M. genitalium antigen preparcd in the same way as the M. pneumoniae antigen (unpublished observations). Obviously, the EIA would not discriminate between human antibodies to the two cross-reacting species (23). Although M. genitaliumn has bcen isolated from throat swabs of patients (1), it is not yet known whether the organism is a human pathogen. In the search for possible M. genitalium infections, we performed immunoblotting with several EIApositive sera against both M. ptnewunoniae and M. genitaliiwn; however, all positivc sera showed reactions with both antigens; a few stronger reactions seen with M. genitalium were inconclusive. We have not considered the possibility of M. genitaliumn infection in the evaluation of the specificity of the EIA. We believe that this EIA, which has a specificity of 99.7%, a sensitivity of 97.8&% compared with the MPCF test, and a relative good reproducibility, is a realistic diagnostic alternative to the MPCF test evcl if it is supplemented with the CA test. REFERENCES 1. Baseman, J. B., S. F. Dallo, J. G. Tully, and D. L. Rose. 1988. Isolation and characterization of Mycoplasma genitaliumn strains from the human respiratory tract. J. Clin. Microbiol.
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