Journal of Infection (I992) 25, Supplement I, 87-9o
P r e v a l e n c e o f a n t i b o d i e s to Chlamydia Belgian population
pneumoniae
in a
A n n e - M a r i e Van den Abeele, Lieve Van R e n t e r g h e m , Kristel W i l l e m s and Jean P l u m
Bacteriology and Virology Laboratory, University Hospital, Block A, De Pintelaan I85, B - 9 o o o Ghent, Belgium Summary T o evaluate the prevalence of antibodies to Chlamydia pneumoniae in a healthy adult Belgian population a study group of I5o medical students was chosen. Sera were collected in the period between March and October I99O and assessed by the microimmunofluorescence test. Sixty-one per cent were found to have IgG antibodies to C. pneumoniae in a titre ~ I6, which showed evidence of past infection. Twenty-one per cent had IgA in a titre ~ 8. In none were antibodies of the IgM fraction detected. T he same sera were tested for the presence of antibodies to Chlamydia trachomatis. One hundred and thirty-one sera with no or low titres of antibodies to C. pneumoniae tended to have low or no detectable antibodies to C. trachomatis. Nineteen sera with high ( > i28) titres of antibodies to C. pneumoniae had IgG antibodies in a titre of 32 to C. trachomatis. This prevalence (I3 %) is much higher than one would expect in a population at low risk for C. trachomatis infection. T he problem of possible crossreactions between the three species in the micro-immunofluorescence test is discussed.
Introduction
Chlamydia pneumoniae is a r e c e nt l y discovered pathogen. 1 It was initially r e g a r d e d as a subspecies o f Chlamydia psittaci. I n I989, h o w e v e r , based on its u n i q u e m o r p h o l o g y , i m m u n o l o g i c a l characteristics and D N A h o m o l o g y it was defined as a new, t h i r d Chlamydia s p e c i e s ) Chlamydia pneumoniae is c o n s i d e r e d as a h u m a n r e s p i r a t o r y p a t h o g e n w i t h o u t a k n o w n animal reservoir. Seroepidemiological studies seem to indicate a w o r l d w i d e d i s t r i b u t i o n o f the a g e n t ) N o t m u c h is k n o w n a b o u t the s e r o p r e v a l e n c e o f C. pneumoniae in B e l g i u m and its s u r r o u n d i n g countries. T o fill in this gap we started car r yi ng out seroprevalence studies in I989. T h e aim o f o u r p r e s e n t s t udy is to evaluate the seroprevalence of C. pneumoniae in a y o u n g , adult p o pul a t i on. Patients and methods Sera o f I5O medical students were collected in the p e r i o d b e t w e e n M a r c h and O c t o b e r I99o. T h e s e s t ude nt s v o l u n t e e r e d to participate in a st udy a b o u t R h i n o v i r u s infection and t he specimens consisted of p r e - s t u d y screenings. F o r d etectio n o f antibodies to C. pneumoniae a modification o f the m i c r o i m m u n o f l u o r e s c e n c e test of W a n g et al. 4 was used. T h e antigen was o b t a i n e d f r o m th e W a s h i n g t o n R e s e a r c h F o u n d a t i o n and was coated on teflon slides with 36 wells o f 2 m m diameter. All sera were screened for total antibodies with a starting di l ut i on o f I / i 6 . F o r titration of I g G , IgA and I g M a starting dilution o f 1/8 was chosen. oI63-4453/92/SIoo87 +04 $03.00/0
© I992 The British Society for the Study of Infection
A.-M. VAN DEN ABEELE ET A L .
88
T a b l e I Comparison of IgG antibody titres to Chlamydia p n e u m o n i a e and Chlamydia trachomatis measured by two immunofluorescence tests C. pneumoniae I g G titre
Chlamydia trachomatis I g G titres
Negative Low ( > 8 - < 32) High ( ~> 3 2)
Negative
Low ( > i 6 - < I28)
High ( > I28)
59
69
o
o
3
o
o
o
I9
F o r C. trachomatis a commercial immunofluorescence test (Biomdrieux, France) was used. In this test slides were coated with antigen from C. trachomatis L2. I g G antibodies were titrated with a starting dilution of 1/8. T h e conjugate was fluorescein labelled sheep anti-human i m m u n o g l o b u l i n (Wellcome, U . K . ) . F o r detection of specific I g G , I g M and IgA appropriate conjugates were used. T h e interpretation of serology was based on the definition of positive results as p r o p o s e d b y G r a y s t o n et al) All m i c r o - i m m u n o f l u o r e s c e n c e tests were p e r f o r m e d b y the same two persons w h o b o t h read the slides separately and c o m p a r e d their results later on. Titres differed b y at most one dilution b e t w e e n the two readers and w h e n this occurred invariably the lowest titre was chosen. Results
First, all sera were tested for the presence of total antibodies to C. pneumoniae. Ninety-five students (63 %) were f o u n d to be positive for total antibodies in a titre ~ 16. T h e s e sera were tested further. N i n e t y - o n e students ( 6 1 % ) had I g G in a titre i> 16. T h i r t y - t h r e e ( 2 1 % ) were f o u n d to possess IgA in a titre >i 8. In none were antibodies of the I g M fraction detected. T h e same 15o sera were tested for the presence of I g G antibodies to C. trachomatis b y the immunofluorescence test. T h e results are s u m m a r i s e d in T a b l e I. F r o m the 59 negative sera for antibodies to C. pneumoniae none was positive for antibodies to C. trachomatis. In the group of 72 students with low levels of antibodies ( > 1 6 - < 128) to C. pneumoniae two had antibodies to C. trachomatis in a titre of 8 and one had a titre of 16. N i n e t e e n sera with a titre of antibodies to C. trachomatis ~ 32 belonged to the group with I g G antibody titres > 128 and IgA > 16 to C. pneumoniae. Discussion
T a b l e I shows the prevalence of antibodies to C. pneumoniae. In early studies I g G antibody titres ~ 32 were considered chronic or p r e - e x i s t i n g ) Recently, G r a y s t o n et al. suggested that a titre > 16 should be used to d e t e r m i n e population prevalence. 3 U n d e r this criterion 6 1 % of our study population
Chlamydial antibodies in Belgians
89
shows evidence of past infection. It has been suggested that sera with IgA antibodies to C. pneumoniae tend to have higher titres of I g G antibodies than those w i t h o u t an IgA titre; however, a positivity limit for IgA antibodies to C. pneumoniae has not yet been established. Saikku et al. ~ reported recently on the possible relationship of chronic chlamydial infection with chronic coronary heart disease and acute myocardial infarction. T h e y state that high, stable IgA titres ( > 32) together with high I g G titres w i t h o u t seroconversion and no I g M antibodies suggest chronic C. pneumoniae infection, b u t the significance and the possible pathological implications of this need further investigation. T h e fact that none of our sera tested showed I g M antibodies suggests that the incidence of n e w cases of C. pneumoniae infection in our study group during the study period was low. W h e n testing the I5 ° sera for evidence of antibodies to C. trachomatis we divided t h e m into three groups according to the height of antibody titre for C. pneumoniae. As s h o w n in T a b l e I, there is a statistically significant correlation b e t w e e n the height of antibody titre for C. pneumoniae vs. C. trachomatis (Chisquare with Yates correction P < o'oo5). Cross-reactivity is the most obvious reason since the s t u d y group is at low risk for C. trachomatis infection and no antibodies against C. trachomatis could be detected in the group of 59 sera negative for C. pneumoniae. T h i s is in agreement with a previous study where we investigated the seroprevalence of antibodies to C. pneumoniae in a paediatric hospital population. 7 W e showed that in this paediatric population, where p r i m a r y infection for C. pneumoniae can be expected, the same degree of cross-reaction could be observed. W a n g and G r a y s t o n 4 are able to read their m i c r o - i m m u n o f l u o r e s c e n c e test with a high degree of specificity for the different species and serovars. We, on the contrary, believe that cross-reactions are inevitable. Firstly, one has to differentiate b e t w e e n two distinct types of fluorescence: a specific and a nonspecific one. s T h e specific fluorescence is related to antibodies directed against a species-specific antigen on the M a j o r O u t e r M e m b r a n e Protein ( M O M P ) . T h i s results in a clear, bright fluorescence m u c h like a starry sky. T h e nonspecific fluorescence is mostly caused b y reaction to a genus specific antigen, the lipopolysaccharide fraction (LPS). T h i s results in a patchy, scattered fluorescence pattern. T o be able to read the test with specificity one has to achieve a certain degree o f experience. Secondly, the M O M P not only exhibits species-specific epitopes b u t also genus-specific ones. 9 A third important remark is that w h e n comparing fluorescence tests one with the other, identical test procedures for the different genera or serovars must be used. This was not the case in our s t u d y where a commercial immunofluorescence test for C. trachomatis was used vs. a modified microimmunofluorescence test for C. pneumoniae. Part of the cross-reaction could be attributed to this. In conclusion, the study shows that the prevalence of antibodies to C. pneumoniae in this young, healthy, adult population is comparable to the one o b s e r v e d b y several other investigators t h r o u g h o u t the world. It is also important to appreciate that in an adult population false-positivity rates of seroprevalence studies of C. trachomatis evaluated b y the microimmunofluorescence test m a y be unacceptably high due to a cross-reaction with C. pneumoniae.
90
A.-M. VAN DEN ABEELE ET A L .
References I. Grayston JT, Kuo CC, Wang SP, Altman J. A new C. psittaci strain, TWAR, isolated in acute respiratory tract infections. N Engl J IVied I986; 3r5 : I6I-I68. 2. Grayston JT, Kuo CC, Campbell LA, Wang SP. Chlamydia pneumoniae sp. nov. for Chlamydia sp. strain TWAR. Int J Syst Bacteriol I989; 39: 88-90. 3. Grayston JT, Wang SP, Kuo CC, Campbell LA. Current knowledge on Chlamydia pneumoniae, strain TWAR, an important cause of pneumonia and other acute respiratory diseases. Eur J Clin Microbiol Infect Dis I99o; 9: 347-3494. Wang SP, Grayston JT. Microimmunofluorescence serological studies with the TWAR organism. In: Oriel JD, Ridgway G, Schachter J, Taylor-Robinson D, Ward M, Eds. Chlamydial infections. Cambridge: Cambridge University Press, I986:329-33 z. 5. Wang SP, Grayston JT. Population prevalence antibody to Chlamydia pneumoniae, strain TWAR. In: Bowie WR, Caldwell HD, Jones RP et al., Eds. Chlamydial infections. Cambridge: Cambridge University Press, I99O: 402-405. 6. Saikku P, Mattila K, Nieminen NS et al. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet I988; ii: 983-986. 7. Van Renterghem L, Van den Abeele AM, Claeys G, Plum J. Prevalence of antibodies to Chlamydia pneumoniae in a pediatric hospital population in Belgium. Eur J Clin Microbiol Infect Dis I99o; 9: 347-3498. Schachter J. Human Chlamydia psittaci infection. In: Oriel JD, Ridgway G, Schachter J, Taylor-Robinson D, Ward M, Eds. Chlamydial infections. Cambridge: Cambridge University Press, I986:311-32o. 9- Allan I. Chlamydial antigenic structure and genetics. In: Oriel JD, Ridgway G, Schachter J, Taylor-Robinson D, Ward M, Eds. Chlamydial infections. Cambridge: Cambridge University Press, I986: 73-76.
P r e v a l e n c e o f a n t i b o d i e s to Chlamydia Belgian population
pneumoniae
in a
A n n e - M a r i e Van den Abeele, Lieve Van R e n t e r g h e m , Kristel W i l l e m s and Jean P l u m
Bacteriology and Virology Laboratory, University Hospital, Block A, De Pintelaan I85, B - 9 o o o Ghent, Belgium Summary T o evaluate the prevalence of antibodies to Chlamydia pneumoniae in a healthy adult Belgian population a study group of I5o medical students was chosen. Sera were collected in the period between March and October I99O and assessed by the microimmunofluorescence test. Sixty-one per cent were found to have IgG antibodies to C. pneumoniae in a titre ~ I6, which showed evidence of past infection. Twenty-one per cent had IgA in a titre ~ 8. In none were antibodies of the IgM fraction detected. T he same sera were tested for the presence of antibodies to Chlamydia trachomatis. One hundred and thirty-one sera with no or low titres of antibodies to C. pneumoniae tended to have low or no detectable antibodies to C. trachomatis. Nineteen sera with high ( > i28) titres of antibodies to C. pneumoniae had IgG antibodies in a titre of 32 to C. trachomatis. This prevalence (I3 %) is much higher than one would expect in a population at low risk for C. trachomatis infection. T he problem of possible crossreactions between the three species in the micro-immunofluorescence test is discussed.
Introduction
Chlamydia pneumoniae is a r e c e nt l y discovered pathogen. 1 It was initially r e g a r d e d as a subspecies o f Chlamydia psittaci. I n I989, h o w e v e r , based on its u n i q u e m o r p h o l o g y , i m m u n o l o g i c a l characteristics and D N A h o m o l o g y it was defined as a new, t h i r d Chlamydia s p e c i e s ) Chlamydia pneumoniae is c o n s i d e r e d as a h u m a n r e s p i r a t o r y p a t h o g e n w i t h o u t a k n o w n animal reservoir. Seroepidemiological studies seem to indicate a w o r l d w i d e d i s t r i b u t i o n o f the a g e n t ) N o t m u c h is k n o w n a b o u t the s e r o p r e v a l e n c e o f C. pneumoniae in B e l g i u m and its s u r r o u n d i n g countries. T o fill in this gap we started car r yi ng out seroprevalence studies in I989. T h e aim o f o u r p r e s e n t s t udy is to evaluate the seroprevalence of C. pneumoniae in a y o u n g , adult p o pul a t i on. Patients and methods Sera o f I5O medical students were collected in the p e r i o d b e t w e e n M a r c h and O c t o b e r I99o. T h e s e s t ude nt s v o l u n t e e r e d to participate in a st udy a b o u t R h i n o v i r u s infection and t he specimens consisted of p r e - s t u d y screenings. F o r d etectio n o f antibodies to C. pneumoniae a modification o f the m i c r o i m m u n o f l u o r e s c e n c e test of W a n g et al. 4 was used. T h e antigen was o b t a i n e d f r o m th e W a s h i n g t o n R e s e a r c h F o u n d a t i o n and was coated on teflon slides with 36 wells o f 2 m m diameter. All sera were screened for total antibodies with a starting di l ut i on o f I / i 6 . F o r titration of I g G , IgA and I g M a starting dilution o f 1/8 was chosen. oI63-4453/92/SIoo87 +04 $03.00/0
© I992 The British Society for the Study of Infection
A.-M. VAN DEN ABEELE ET A L .
88
T a b l e I Comparison of IgG antibody titres to Chlamydia p n e u m o n i a e and Chlamydia trachomatis measured by two immunofluorescence tests C. pneumoniae I g G titre
Chlamydia trachomatis I g G titres
Negative Low ( > 8 - < 32) High ( ~> 3 2)
Negative
Low ( > i 6 - < I28)
High ( > I28)
59
69
o
o
3
o
o
o
I9
F o r C. trachomatis a commercial immunofluorescence test (Biomdrieux, France) was used. In this test slides were coated with antigen from C. trachomatis L2. I g G antibodies were titrated with a starting dilution of 1/8. T h e conjugate was fluorescein labelled sheep anti-human i m m u n o g l o b u l i n (Wellcome, U . K . ) . F o r detection of specific I g G , I g M and IgA appropriate conjugates were used. T h e interpretation of serology was based on the definition of positive results as p r o p o s e d b y G r a y s t o n et al) All m i c r o - i m m u n o f l u o r e s c e n c e tests were p e r f o r m e d b y the same two persons w h o b o t h read the slides separately and c o m p a r e d their results later on. Titres differed b y at most one dilution b e t w e e n the two readers and w h e n this occurred invariably the lowest titre was chosen. Results
First, all sera were tested for the presence of total antibodies to C. pneumoniae. Ninety-five students (63 %) were f o u n d to be positive for total antibodies in a titre ~ 16. T h e s e sera were tested further. N i n e t y - o n e students ( 6 1 % ) had I g G in a titre i> 16. T h i r t y - t h r e e ( 2 1 % ) were f o u n d to possess IgA in a titre >i 8. In none were antibodies of the I g M fraction detected. T h e same 15o sera were tested for the presence of I g G antibodies to C. trachomatis b y the immunofluorescence test. T h e results are s u m m a r i s e d in T a b l e I. F r o m the 59 negative sera for antibodies to C. pneumoniae none was positive for antibodies to C. trachomatis. In the group of 72 students with low levels of antibodies ( > 1 6 - < 128) to C. pneumoniae two had antibodies to C. trachomatis in a titre of 8 and one had a titre of 16. N i n e t e e n sera with a titre of antibodies to C. trachomatis ~ 32 belonged to the group with I g G antibody titres > 128 and IgA > 16 to C. pneumoniae. Discussion
T a b l e I shows the prevalence of antibodies to C. pneumoniae. In early studies I g G antibody titres ~ 32 were considered chronic or p r e - e x i s t i n g ) Recently, G r a y s t o n et al. suggested that a titre > 16 should be used to d e t e r m i n e population prevalence. 3 U n d e r this criterion 6 1 % of our study population
Chlamydial antibodies in Belgians
89
shows evidence of past infection. It has been suggested that sera with IgA antibodies to C. pneumoniae tend to have higher titres of I g G antibodies than those w i t h o u t an IgA titre; however, a positivity limit for IgA antibodies to C. pneumoniae has not yet been established. Saikku et al. ~ reported recently on the possible relationship of chronic chlamydial infection with chronic coronary heart disease and acute myocardial infarction. T h e y state that high, stable IgA titres ( > 32) together with high I g G titres w i t h o u t seroconversion and no I g M antibodies suggest chronic C. pneumoniae infection, b u t the significance and the possible pathological implications of this need further investigation. T h e fact that none of our sera tested showed I g M antibodies suggests that the incidence of n e w cases of C. pneumoniae infection in our study group during the study period was low. W h e n testing the I5 ° sera for evidence of antibodies to C. trachomatis we divided t h e m into three groups according to the height of antibody titre for C. pneumoniae. As s h o w n in T a b l e I, there is a statistically significant correlation b e t w e e n the height of antibody titre for C. pneumoniae vs. C. trachomatis (Chisquare with Yates correction P < o'oo5). Cross-reactivity is the most obvious reason since the s t u d y group is at low risk for C. trachomatis infection and no antibodies against C. trachomatis could be detected in the group of 59 sera negative for C. pneumoniae. T h i s is in agreement with a previous study where we investigated the seroprevalence of antibodies to C. pneumoniae in a paediatric hospital population. 7 W e showed that in this paediatric population, where p r i m a r y infection for C. pneumoniae can be expected, the same degree of cross-reaction could be observed. W a n g and G r a y s t o n 4 are able to read their m i c r o - i m m u n o f l u o r e s c e n c e test with a high degree of specificity for the different species and serovars. We, on the contrary, believe that cross-reactions are inevitable. Firstly, one has to differentiate b e t w e e n two distinct types of fluorescence: a specific and a nonspecific one. s T h e specific fluorescence is related to antibodies directed against a species-specific antigen on the M a j o r O u t e r M e m b r a n e Protein ( M O M P ) . T h i s results in a clear, bright fluorescence m u c h like a starry sky. T h e nonspecific fluorescence is mostly caused b y reaction to a genus specific antigen, the lipopolysaccharide fraction (LPS). T h i s results in a patchy, scattered fluorescence pattern. T o be able to read the test with specificity one has to achieve a certain degree o f experience. Secondly, the M O M P not only exhibits species-specific epitopes b u t also genus-specific ones. 9 A third important remark is that w h e n comparing fluorescence tests one with the other, identical test procedures for the different genera or serovars must be used. This was not the case in our s t u d y where a commercial immunofluorescence test for C. trachomatis was used vs. a modified microimmunofluorescence test for C. pneumoniae. Part of the cross-reaction could be attributed to this. In conclusion, the study shows that the prevalence of antibodies to C. pneumoniae in this young, healthy, adult population is comparable to the one o b s e r v e d b y several other investigators t h r o u g h o u t the world. It is also important to appreciate that in an adult population false-positivity rates of seroprevalence studies of C. trachomatis evaluated b y the microimmunofluorescence test m a y be unacceptably high due to a cross-reaction with C. pneumoniae.
90
A.-M. VAN DEN ABEELE ET A L .
References I. Grayston JT, Kuo CC, Wang SP, Altman J. A new C. psittaci strain, TWAR, isolated in acute respiratory tract infections. N Engl J IVied I986; 3r5 : I6I-I68. 2. Grayston JT, Kuo CC, Campbell LA, Wang SP. Chlamydia pneumoniae sp. nov. for Chlamydia sp. strain TWAR. Int J Syst Bacteriol I989; 39: 88-90. 3. Grayston JT, Wang SP, Kuo CC, Campbell LA. Current knowledge on Chlamydia pneumoniae, strain TWAR, an important cause of pneumonia and other acute respiratory diseases. Eur J Clin Microbiol Infect Dis I99o; 9: 347-3494. Wang SP, Grayston JT. Microimmunofluorescence serological studies with the TWAR organism. In: Oriel JD, Ridgway G, Schachter J, Taylor-Robinson D, Ward M, Eds. Chlamydial infections. Cambridge: Cambridge University Press, I986:329-33 z. 5. Wang SP, Grayston JT. Population prevalence antibody to Chlamydia pneumoniae, strain TWAR. In: Bowie WR, Caldwell HD, Jones RP et al., Eds. Chlamydial infections. Cambridge: Cambridge University Press, I99O: 402-405. 6. Saikku P, Mattila K, Nieminen NS et al. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet I988; ii: 983-986. 7. Van Renterghem L, Van den Abeele AM, Claeys G, Plum J. Prevalence of antibodies to Chlamydia pneumoniae in a pediatric hospital population in Belgium. Eur J Clin Microbiol Infect Dis I99o; 9: 347-3498. Schachter J. Human Chlamydia psittaci infection. In: Oriel JD, Ridgway G, Schachter J, Taylor-Robinson D, Ward M, Eds. Chlamydial infections. Cambridge: Cambridge University Press, I986:311-32o. 9- Allan I. Chlamydial antigenic structure and genetics. In: Oriel JD, Ridgway G, Schachter J, Taylor-Robinson D, Ward M, Eds. Chlamydial infections. Cambridge: Cambridge University Press, I986: 73-76.
