Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
NOTES
solid state 13cNMR and FT-IR: bacteriorhodopsin test case. In preparation. Kinsey, R.A., Kintanar, A., and Oldfield, E. 1981. Dynamics of amino acid side chains in membrane proteins by high field solid state NMR spectroscopy. J . Biol. Chem. 256: 9028-9036. Lanyi, J.K. 1986. Bacteriorhodopsin and related light-energy converters. Bioenergetics. Edited by L. Ernster. Elsevier Science Publishers B.V., Amsterdam. pp. 3 15-350. Lewis, B.A., Harbison, G.S., Herzfeld, J., and Griffin, R.G. 1985. NMR structural analysis of a membrane protein: bacteriorhodopsin peptide backbone orientation and motion. Biochemistry, 24: 467 1-4679. Oesterhelt, D., and Stoeckenius, W. 1974. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 31: 667-678. Onishi, H., McCance, M.E., and Gibbons, N.E. 1965. A synthetic
medium for extremely halophilic bacteria. Can. J. Microbiol. 11: 365-373. Ross, P.E., Helgerson, S.L., Miercke, L.J., and Dratz, E.A. 1989. Isoelectric focusing studies of bacteriorhodopsin. Biochim. Biophys. Acta, 991: 134-140. Signeuret, M., Neumann, J.-M., Levy, D., and Rigaud, J.-L. 1991. High-resolution 13C NMR study of the topography and dynamics of methionine residues in detergent solubilized bacteriorhodopsin. Biochemistry, 30: 3885-3892. Stoeckenius, W., and Bogomolni, R.A. 1982. Bacteriorhodopsin and related pigments in halobacteria. Annu. Rev. Biochem. 52: 587-616. Weber, H . J., and Bogomolni, R.A. 1982. The isolation of halobacterium mutant strains with defects in pigment synthesis. Methods Enzymol. 88: 379-390.
Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections GERARD OZANNE
' AND JOHANNELEFEBVRE
Laboratoire de santk publique du Qukbec, Immunodiagnostic and Chlamydia, 20045 chemin Ste-Marie, Ste-Anne-de-Bellevue, Qutbec, Canada H 9 X 3R5 Received February 14, 1992 Revision received June 25, 1992 Accepted June 29, 1992 OZANNE,G., and LEFEBVRE,J. 1992. Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections. Can. J. Microbiol. 38: 1185-1 189. Chlamydia pneumoniae infections are mostly confirmed using an indirect microimmunofluorescence test for which potential cross-reactions between antigens from different chlamydial species are not well documented. Using this assay, 928 sera (507 subjects) submitted for Chlamydia pneumoniae serology were tested for specific IgM and IgG to this bacteria using the TW-183 antigen. IgM and IgG reactivities t o Chlamydia trachomatis serotypes C , D, E, and L2 and Chlamydia psittaci strain 6BC antigens were also tested. A sample was interpreted as positive only when evenly fluorescent elementary bodies were observed. Twenty-five subjects (4.9%) showed serological evidence of recent Chlamydia pneumoniae infection (IgM positive and (or) IgG seroconversion); 11 of them also showed serological evidence of recent infection with at least one other chlamydial species. Specificity was 50 and 63% for IgM and IgG detection, respectively. These results suggest that mixed or temporally related infections might occur, or, more likely, that some Chlamydia pneumoniae IgM or IgG reactivities might be due t o heterotypic antibodies. Key words: TWAR serology, TWAR infections, TWAR cross-reactions. OZANNE,G., et LEFEBVRE,J. 1992. Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections. Can. J. Microbiol. 38 : 1185- 1189. Les infections a Chlamydia pneumoniae sont habituellement confirmees par une methode de microimmunofluorescence indirecte pour laquelle on connaft peu les possibilites de reactions croisees entre les antigenes des diverses especes de Chlamydia. Un Cchantillon de 928 serums (507 sujets) reGus pour une serologie Chlamydia pneumoniae, a ete teste par microimmunofluorescence, en utilisant l'antigene TW-183, pour verifier la presence d'IgM et d'IgG specifiques a cette bactirie. On a aussi verifie la reactivite des IgM et des IgG avec les antigenes des serotypes C , D, E et L2 de Chlamydia trachomatis et de la souche 6BC de Chlamydia psittaci. Un echantillon etait considere comme positif uniquement lorsque les corps elementaires etaient uniformement fluorescents. Vingt-cinq sujets (4,9%) presentaient une serologie confirmant une infection recente a Chlamydia pneumoniae (presence d'IgM et (ou) seroconversion IgG); 11 d'entre eux presentaient une preuve serologique d'une infection recente par au moins une autre espece de Chlamydia. La specificite de detection des IgM et des IgG etait de 50 et 63% respectivement. Ces resultats laissent croire que des infections mixtes ou temporellement reliees peuvent survenir ou, ce qui semble le plus probable, que certaines reactivitees IgM ou IgG dirigees contre Chlamydia pneumoniae soient dues a des anticorps heterotypiques. Mots clks : serologie TWAR, infections TWAR, reactions croisees TWAR. [Traduit par la redaction] ' ~ u t h o rt o whom all correspondence should be addressed. Printed in Canada / lmprime au Canada
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CAN. J. MICROBIOL. VOL. 38, 1992
Chlamydia pneumoniae (TWAR) bacterial strains have been associated with acute respiratory disease (Grayston et al. 1986; Mordhorst et al. 1986; Chirgwin et al. 1991), coronary heart disease (Saikku et al. 1988), and otitis media with effusion (Ogawa et al. 1990). Isolation of TWAR organisms from clinical samples is difficult (Mardh et al. 1989). Thus, infections are usually diagnosed using serological tests to detect a recent immune response to TWAR antigen (Schachter 1991). The microimmunofluorescence assay (MIF) is the most frequently used (Black et al. 1991; Wang and Grayston 1970). In 1988, we reported the results from a retrospective study which suggested that TWAR infections occurred in Quebec (Ozanne et al. 1988) and that cross-reactions with C. trachomatis serotype D and C. psittaci strain 6BC were uncommon (Ozanne and Lefebvre 1989). However, later a greater proportion of cross-reactions was observed in our laboratory following the introduction of a larger panel of C. trachomatis antigens to control for TWAR antigen specificity. Even though some authors have reported that, in their laboratories, cross-reactions are uncommon (Wang and Grayston 1986; Grayston et al. 1989), other authors have reported several occurrences of cross-reactions between antibody against TWAR and C. psittaci or C. trachomatis antigens (Black et al. 1991; Bourke et al. 1989; Erntell et al. 1989; Persson and Treharne 1989; Van Renterghem et al. 1990; Wreghitt et al. 1990). These contradictory reports and the cross-reactions observed in our laboratory led us to study the immunological specificity (Berzofsky and Schechter 1981) of the TWAR antigen to detect specific IgM and IgG when using MIF for the serodiagnosis of TWAR infections. This study of a large panel of paired human sera showed that some subjects with serological evidence of recent TWAR infections also showed evidence of other recent chlamydial infections, suggesting that some TWAR MIF reactivities might be due to heterotypic antibodies. TWAR MIF was performed on 928 sera (507 subjects) received from June 13, 1988, to April 1, 1991, at the Laboratoire de sante publique du Quebec. All these sera were submitted specifically for TWAR serology. Culture was not possible at the time. Average time lapse between first and second serum was 19 days (mode, 15 days; median, 15 days with a range of 5-45 days). Date of onset of symptoms was not documented for most of the cases. Age of the subjects ranged from 3 months to 96 years (mean, 43 years; median, 40 years; mode, 38 years). Females and males were equally represented. Anti-Chlamydia IgM and IgG were detected using a MIF assay (Wang and Grayston 1970) with the appropriate conjugate. Antigens were TWAR strain TW-183 (Washington Research Foundation, Washington), a pool of C. trachomatis antigens comprising serotype C (strain TW-3/0T), D (strain UW-3/Cx), E (strain UW-5/Cx), and L2 (strain 434/Bu) (Laboratory Center for Disease Control (LCDC), Ottawa, Ontario), C. psittaci strain 6BC (American type culture collection ATCC VR- 125). Conjugates were either fluorescein isothiocyanate (FITC) labeled goat anti-human IgG (Sigma Chemical Co., St. Louis, Missouri) or FITC-labeled sheep anti-human IgM (Jackson Immuno Research, BIO/CAN Scientific, Mississauga, Ontario). Both conjugates were used at an optimal box titration working dilution of 1:80. Reading was carried out using a Dialux microscope (Leitz) equipped
with an HBO 50 lamp, KP500 exciter filter, K510-530 barrier filter, 10X ocular, and 40/0.65 E F objective. Serum starting dilution was 1:8 for all antigens. A sample was interpreted as positive when an evenly distributed fluorescence associated only with the elementary bodies was observed. Serological reactions suggesting current TWAR infection were either a fourfold IgG antibody titer increase or decrease, specific IgM antibody titer 2 1: 16, or a specific IgG antibody titer 2 1:5 12 (Grayston et al. 1989; Thom et al. 1990). Serological result indicating past TWAR infection was a stable specific IgG titer between 1: 16 and 1:256 (Grayston et al. 1989). For the other antigens, titers were recorded without interpretation. To calculate antigen specificity, only serological responses suggesting a recent immunogenic contact were considered. Cross-reactivity was evaluated using contingency tables. TWAR antigen specificity was calculated by dividing the number of subjects showing significant reactivity only against TWAR antigen by the number of subjects showing TWAR reactivity alone or with other chlamydial reactivity. Specific IgG at a stable titer below 1:5 12 (past infection) were detected in 80 subject (16%). Twenty-five subjects (4.9%) showed serological evidence of recent TWAR immunogenic contact. They were distributed as follows: 9/507 subjects (1.8%) were anti-TWAR IgM positive (Table l), 16/507 subjects (3.1 %) showed either a fourfold increase or decrease in anti-TWAR IgG titer or an IgG titer 2512 without specific IgM (Table 2). Five IgM positive subjects (Table 1, ID Nos. 1, 2, 3, 4, and 5) also showed an IgG immune response compatible with a recent TWAR immunogenic contact. To evaluate the specificity of the TWAR IgM reactivity, only concurrent IgM reactivities (titer 1 1: 16) against different chlamydial antigens were studied. Among the eight anti-TWAR IgM positive subjects tested for other IgM reactivities (Table l), three subjects (ID Nos. 1, 5, and 7) showed IgM reacting with the three chlamydial antigens and one subject (ID No. 8) showed IgM reacting with Chlamydia trachomatis antigen giving a specificity of 50% (4/8) for IgM MIF reactivity when using TWAR (strain TW-183) antigen. To evaluate the specificity of the TWAR IgG reactivity, only concurrent significant IgG responses against different chlamydial antigens were studied. Among the 2 1 subjects showing significant anti-TWAR IgG response, 19 were tested for other IgG reactivities (Tables 1 and 2). Concurrent IgG seroconversions were observed for the three chlamydial antigens in one subject (ID No. 1) and for TWAR and C . psittaci antigen in another subject (ID No. 14). One subject (ID No. 11) had an IgG titer 2 1:512 for all three chlamydial antigens. One subject (ID No. 24) showed an IgG titer decrease for both TWAR and C. trachomatis. One subject (ID No. 10) showed an anti-TWAR IgG titer >1:1024 along with an IgG seroconversion to both C . trachomatis and C . psittaci. One subject (ID No. 18) showed an anti-TWAR IgG seroconversion and a significant decrease in anti-C. trachomatis IgG. Finally, one subject (ID No. 20) showed an anti-TWAR IgG seroconversion and an IgG titer 2 1:512 for the other two chlamydial species. Antigenic specificity for the detection of a significant IgG response was 63% (12/29) using TWAR (strain TW-183) antigen. Finally, to obtain the overall specificity of the TWAR antigen in MIF, both concurrent IgM and IgG reactivities
1187
NOTES
TABLE1 . Subjects with Chlamydia pneumoniae IgM positive sera
TABLE2. Subjects showing only IgG serological evidence of Chlamydia pneumoniae recent immunogenic contact
Microimmunofluorescence titers Microimmunofluorescence IgG titers
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
Subject ID
Days between sera
Chlamydia pneumoniae IgM
IgG
Chlamydia trachomatis IgM
IgG
Chlamydia psittaci IgM
Subject ID
Days between sera
Chlamydia pneumoniae
Chlamydia trachomatis
Chlamydia psittaci
IgG
NOTE: For each subject, the first line shows serologic results of the first serum and the second line shows results of the second serum. Sera of subjects 1-5 are IgM and IgG reactive. Subjects 1-3 are pneumonia cases, subject 4 is a bronchitis case, the clinical status of subjects 5-8 is not documented, and subject 9 is a myocarditis case. *Serum not reactive at the cutoff point. + ~ odone t (insufficient volume).
against different antigens were considered. Two subjects (ID Nos. 6 and 10) showed IgG and IgM reactivities against several chlamydial antigens. Overall, 11/22 subjects (ID Nos. 1, 5, 6, 7, 8, 10, 11, 14, 18, 20, and 24) had sera showing multiple significant reactivities giving a TWAR antigen specificity of 50% for confirmation of recent TWAR immunogenic contact using MIF. The possibility of cross-reactions when performing TWAR MIF is not well documented (Black et al. 1991), previous reports about the specificity of the test being contradictory (Bourke et al. 1989; Grayston et al. 1986, 1989; Persson and Treharne 1989; Schachter 1986; Van Renterghem et al. 1990; Wang and Grayston 1986; Wreghitt et al. 1990). In our study, 50070 of the subjects showing serological evidence of recent immunogenic contact with TWAR also showed a significant reactivity to at least one other chlamydial species (Tables 1 and 2), indicating that MIF might not be as discriminating as previously reported by some authors (Grayston et al. 1989; Wang and Grayston 1986). In MIF, the immunodominant antigen is probably the major outer membrane protein (MOMP) (Saikku et al. 1988), which is an important surface antigen of both chlamydial elementary and reticulate bodies. MOMP, which displays genus-, species-, subspecies-, and serovar-specific antigen determinants that are recognized during human
19
30
25 12 25
128
NOTES
solid state 13cNMR and FT-IR: bacteriorhodopsin test case. In preparation. Kinsey, R.A., Kintanar, A., and Oldfield, E. 1981. Dynamics of amino acid side chains in membrane proteins by high field solid state NMR spectroscopy. J . Biol. Chem. 256: 9028-9036. Lanyi, J.K. 1986. Bacteriorhodopsin and related light-energy converters. Bioenergetics. Edited by L. Ernster. Elsevier Science Publishers B.V., Amsterdam. pp. 3 15-350. Lewis, B.A., Harbison, G.S., Herzfeld, J., and Griffin, R.G. 1985. NMR structural analysis of a membrane protein: bacteriorhodopsin peptide backbone orientation and motion. Biochemistry, 24: 467 1-4679. Oesterhelt, D., and Stoeckenius, W. 1974. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 31: 667-678. Onishi, H., McCance, M.E., and Gibbons, N.E. 1965. A synthetic
medium for extremely halophilic bacteria. Can. J. Microbiol. 11: 365-373. Ross, P.E., Helgerson, S.L., Miercke, L.J., and Dratz, E.A. 1989. Isoelectric focusing studies of bacteriorhodopsin. Biochim. Biophys. Acta, 991: 134-140. Signeuret, M., Neumann, J.-M., Levy, D., and Rigaud, J.-L. 1991. High-resolution 13C NMR study of the topography and dynamics of methionine residues in detergent solubilized bacteriorhodopsin. Biochemistry, 30: 3885-3892. Stoeckenius, W., and Bogomolni, R.A. 1982. Bacteriorhodopsin and related pigments in halobacteria. Annu. Rev. Biochem. 52: 587-616. Weber, H . J., and Bogomolni, R.A. 1982. The isolation of halobacterium mutant strains with defects in pigment synthesis. Methods Enzymol. 88: 379-390.
Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections GERARD OZANNE
' AND JOHANNELEFEBVRE
Laboratoire de santk publique du Qukbec, Immunodiagnostic and Chlamydia, 20045 chemin Ste-Marie, Ste-Anne-de-Bellevue, Qutbec, Canada H 9 X 3R5 Received February 14, 1992 Revision received June 25, 1992 Accepted June 29, 1992 OZANNE,G., and LEFEBVRE,J. 1992. Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections. Can. J. Microbiol. 38: 1185-1 189. Chlamydia pneumoniae infections are mostly confirmed using an indirect microimmunofluorescence test for which potential cross-reactions between antigens from different chlamydial species are not well documented. Using this assay, 928 sera (507 subjects) submitted for Chlamydia pneumoniae serology were tested for specific IgM and IgG to this bacteria using the TW-183 antigen. IgM and IgG reactivities t o Chlamydia trachomatis serotypes C , D, E, and L2 and Chlamydia psittaci strain 6BC antigens were also tested. A sample was interpreted as positive only when evenly fluorescent elementary bodies were observed. Twenty-five subjects (4.9%) showed serological evidence of recent Chlamydia pneumoniae infection (IgM positive and (or) IgG seroconversion); 11 of them also showed serological evidence of recent infection with at least one other chlamydial species. Specificity was 50 and 63% for IgM and IgG detection, respectively. These results suggest that mixed or temporally related infections might occur, or, more likely, that some Chlamydia pneumoniae IgM or IgG reactivities might be due t o heterotypic antibodies. Key words: TWAR serology, TWAR infections, TWAR cross-reactions. OZANNE,G., et LEFEBVRE,J. 1992. Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections. Can. J. Microbiol. 38 : 1185- 1189. Les infections a Chlamydia pneumoniae sont habituellement confirmees par une methode de microimmunofluorescence indirecte pour laquelle on connaft peu les possibilites de reactions croisees entre les antigenes des diverses especes de Chlamydia. Un Cchantillon de 928 serums (507 sujets) reGus pour une serologie Chlamydia pneumoniae, a ete teste par microimmunofluorescence, en utilisant l'antigene TW-183, pour verifier la presence d'IgM et d'IgG specifiques a cette bactirie. On a aussi verifie la reactivite des IgM et des IgG avec les antigenes des serotypes C , D, E et L2 de Chlamydia trachomatis et de la souche 6BC de Chlamydia psittaci. Un echantillon etait considere comme positif uniquement lorsque les corps elementaires etaient uniformement fluorescents. Vingt-cinq sujets (4,9%) presentaient une serologie confirmant une infection recente a Chlamydia pneumoniae (presence d'IgM et (ou) seroconversion IgG); 11 d'entre eux presentaient une preuve serologique d'une infection recente par au moins une autre espece de Chlamydia. La specificite de detection des IgM et des IgG etait de 50 et 63% respectivement. Ces resultats laissent croire que des infections mixtes ou temporellement reliees peuvent survenir ou, ce qui semble le plus probable, que certaines reactivitees IgM ou IgG dirigees contre Chlamydia pneumoniae soient dues a des anticorps heterotypiques. Mots clks : serologie TWAR, infections TWAR, reactions croisees TWAR. [Traduit par la redaction] ' ~ u t h o rt o whom all correspondence should be addressed. Printed in Canada / lmprime au Canada
1185
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
1186
CAN. J. MICROBIOL. VOL. 38, 1992
Chlamydia pneumoniae (TWAR) bacterial strains have been associated with acute respiratory disease (Grayston et al. 1986; Mordhorst et al. 1986; Chirgwin et al. 1991), coronary heart disease (Saikku et al. 1988), and otitis media with effusion (Ogawa et al. 1990). Isolation of TWAR organisms from clinical samples is difficult (Mardh et al. 1989). Thus, infections are usually diagnosed using serological tests to detect a recent immune response to TWAR antigen (Schachter 1991). The microimmunofluorescence assay (MIF) is the most frequently used (Black et al. 1991; Wang and Grayston 1970). In 1988, we reported the results from a retrospective study which suggested that TWAR infections occurred in Quebec (Ozanne et al. 1988) and that cross-reactions with C. trachomatis serotype D and C. psittaci strain 6BC were uncommon (Ozanne and Lefebvre 1989). However, later a greater proportion of cross-reactions was observed in our laboratory following the introduction of a larger panel of C. trachomatis antigens to control for TWAR antigen specificity. Even though some authors have reported that, in their laboratories, cross-reactions are uncommon (Wang and Grayston 1986; Grayston et al. 1989), other authors have reported several occurrences of cross-reactions between antibody against TWAR and C. psittaci or C. trachomatis antigens (Black et al. 1991; Bourke et al. 1989; Erntell et al. 1989; Persson and Treharne 1989; Van Renterghem et al. 1990; Wreghitt et al. 1990). These contradictory reports and the cross-reactions observed in our laboratory led us to study the immunological specificity (Berzofsky and Schechter 1981) of the TWAR antigen to detect specific IgM and IgG when using MIF for the serodiagnosis of TWAR infections. This study of a large panel of paired human sera showed that some subjects with serological evidence of recent TWAR infections also showed evidence of other recent chlamydial infections, suggesting that some TWAR MIF reactivities might be due to heterotypic antibodies. TWAR MIF was performed on 928 sera (507 subjects) received from June 13, 1988, to April 1, 1991, at the Laboratoire de sante publique du Quebec. All these sera were submitted specifically for TWAR serology. Culture was not possible at the time. Average time lapse between first and second serum was 19 days (mode, 15 days; median, 15 days with a range of 5-45 days). Date of onset of symptoms was not documented for most of the cases. Age of the subjects ranged from 3 months to 96 years (mean, 43 years; median, 40 years; mode, 38 years). Females and males were equally represented. Anti-Chlamydia IgM and IgG were detected using a MIF assay (Wang and Grayston 1970) with the appropriate conjugate. Antigens were TWAR strain TW-183 (Washington Research Foundation, Washington), a pool of C. trachomatis antigens comprising serotype C (strain TW-3/0T), D (strain UW-3/Cx), E (strain UW-5/Cx), and L2 (strain 434/Bu) (Laboratory Center for Disease Control (LCDC), Ottawa, Ontario), C. psittaci strain 6BC (American type culture collection ATCC VR- 125). Conjugates were either fluorescein isothiocyanate (FITC) labeled goat anti-human IgG (Sigma Chemical Co., St. Louis, Missouri) or FITC-labeled sheep anti-human IgM (Jackson Immuno Research, BIO/CAN Scientific, Mississauga, Ontario). Both conjugates were used at an optimal box titration working dilution of 1:80. Reading was carried out using a Dialux microscope (Leitz) equipped
with an HBO 50 lamp, KP500 exciter filter, K510-530 barrier filter, 10X ocular, and 40/0.65 E F objective. Serum starting dilution was 1:8 for all antigens. A sample was interpreted as positive when an evenly distributed fluorescence associated only with the elementary bodies was observed. Serological reactions suggesting current TWAR infection were either a fourfold IgG antibody titer increase or decrease, specific IgM antibody titer 2 1: 16, or a specific IgG antibody titer 2 1:5 12 (Grayston et al. 1989; Thom et al. 1990). Serological result indicating past TWAR infection was a stable specific IgG titer between 1: 16 and 1:256 (Grayston et al. 1989). For the other antigens, titers were recorded without interpretation. To calculate antigen specificity, only serological responses suggesting a recent immunogenic contact were considered. Cross-reactivity was evaluated using contingency tables. TWAR antigen specificity was calculated by dividing the number of subjects showing significant reactivity only against TWAR antigen by the number of subjects showing TWAR reactivity alone or with other chlamydial reactivity. Specific IgG at a stable titer below 1:5 12 (past infection) were detected in 80 subject (16%). Twenty-five subjects (4.9%) showed serological evidence of recent TWAR immunogenic contact. They were distributed as follows: 9/507 subjects (1.8%) were anti-TWAR IgM positive (Table l), 16/507 subjects (3.1 %) showed either a fourfold increase or decrease in anti-TWAR IgG titer or an IgG titer 2512 without specific IgM (Table 2). Five IgM positive subjects (Table 1, ID Nos. 1, 2, 3, 4, and 5) also showed an IgG immune response compatible with a recent TWAR immunogenic contact. To evaluate the specificity of the TWAR IgM reactivity, only concurrent IgM reactivities (titer 1 1: 16) against different chlamydial antigens were studied. Among the eight anti-TWAR IgM positive subjects tested for other IgM reactivities (Table l), three subjects (ID Nos. 1, 5, and 7) showed IgM reacting with the three chlamydial antigens and one subject (ID No. 8) showed IgM reacting with Chlamydia trachomatis antigen giving a specificity of 50% (4/8) for IgM MIF reactivity when using TWAR (strain TW-183) antigen. To evaluate the specificity of the TWAR IgG reactivity, only concurrent significant IgG responses against different chlamydial antigens were studied. Among the 2 1 subjects showing significant anti-TWAR IgG response, 19 were tested for other IgG reactivities (Tables 1 and 2). Concurrent IgG seroconversions were observed for the three chlamydial antigens in one subject (ID No. 1) and for TWAR and C . psittaci antigen in another subject (ID No. 14). One subject (ID No. 11) had an IgG titer 2 1:512 for all three chlamydial antigens. One subject (ID No. 24) showed an IgG titer decrease for both TWAR and C. trachomatis. One subject (ID No. 10) showed an anti-TWAR IgG titer >1:1024 along with an IgG seroconversion to both C . trachomatis and C . psittaci. One subject (ID No. 18) showed an anti-TWAR IgG seroconversion and a significant decrease in anti-C. trachomatis IgG. Finally, one subject (ID No. 20) showed an anti-TWAR IgG seroconversion and an IgG titer 2 1:512 for the other two chlamydial species. Antigenic specificity for the detection of a significant IgG response was 63% (12/29) using TWAR (strain TW-183) antigen. Finally, to obtain the overall specificity of the TWAR antigen in MIF, both concurrent IgM and IgG reactivities
1187
NOTES
TABLE1 . Subjects with Chlamydia pneumoniae IgM positive sera
TABLE2. Subjects showing only IgG serological evidence of Chlamydia pneumoniae recent immunogenic contact
Microimmunofluorescence titers Microimmunofluorescence IgG titers
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
Subject ID
Days between sera
Chlamydia pneumoniae IgM
IgG
Chlamydia trachomatis IgM
IgG
Chlamydia psittaci IgM
Subject ID
Days between sera
Chlamydia pneumoniae
Chlamydia trachomatis
Chlamydia psittaci
IgG
NOTE: For each subject, the first line shows serologic results of the first serum and the second line shows results of the second serum. Sera of subjects 1-5 are IgM and IgG reactive. Subjects 1-3 are pneumonia cases, subject 4 is a bronchitis case, the clinical status of subjects 5-8 is not documented, and subject 9 is a myocarditis case. *Serum not reactive at the cutoff point. + ~ odone t (insufficient volume).
against different antigens were considered. Two subjects (ID Nos. 6 and 10) showed IgG and IgM reactivities against several chlamydial antigens. Overall, 11/22 subjects (ID Nos. 1, 5, 6, 7, 8, 10, 11, 14, 18, 20, and 24) had sera showing multiple significant reactivities giving a TWAR antigen specificity of 50% for confirmation of recent TWAR immunogenic contact using MIF. The possibility of cross-reactions when performing TWAR MIF is not well documented (Black et al. 1991), previous reports about the specificity of the test being contradictory (Bourke et al. 1989; Grayston et al. 1986, 1989; Persson and Treharne 1989; Schachter 1986; Van Renterghem et al. 1990; Wang and Grayston 1986; Wreghitt et al. 1990). In our study, 50070 of the subjects showing serological evidence of recent immunogenic contact with TWAR also showed a significant reactivity to at least one other chlamydial species (Tables 1 and 2), indicating that MIF might not be as discriminating as previously reported by some authors (Grayston et al. 1989; Wang and Grayston 1986). In MIF, the immunodominant antigen is probably the major outer membrane protein (MOMP) (Saikku et al. 1988), which is an important surface antigen of both chlamydial elementary and reticulate bodies. MOMP, which displays genus-, species-, subspecies-, and serovar-specific antigen determinants that are recognized during human
19
30
25 12 25
128
