Pekka Saikku

SEROLOGICAL EVIDENCE OF AN ASSOCIATION OF A NOVEL CHLAMYDIA, TWAR, WITH CHRONIC CORONARY HEART DISEASE AND ACUTE MYOCARDIAL INFARCTION

Paired sera from 40 male patients with acute myocardial infarction (AMI), 30 male patients with chronic coronary heart disease (CCHD), and 41 controls, matched for sex, age, time, and locality were investigated for antibodies to a novel type of Chlamydia sp, TWAR, and to chlamydial lipopolysaccharide (LPS) group antigen. 27 patients with AMI (68%), and 15 (50%) patients with CCHD had raised IgG (greater than or equal to 128) and/or IgA (greater than or equal to 32) titres in the microimmunofluorescence test with chlamydia TWAR. Both frequencies were significantly higher than in the controls (7, 17%). 26 (68%) of 38 patients with AMI also showed a significant seroconversion in enzyme immunoassay with LPS antigen; this response was absent in all patients with CCHD and all but 1 of the controls. Chronic chlamydial infection could be a factor in the pathogenesis of cardiovascular diseases.

Chlamydia pneumoniae-specific circulating immune complexes in patients with chronic coronary heart disease.

Background. An association of chronic Chlamydia pneumoniae infection to coronary heart disease has been suggested recently. In a recent study, we demonstrated circulating immune complexes containing chlamydial genus‐specific lipopolysaccharide in patients with coronary heart disease. The objective of the present study was to investigate whether C. pneumoniae species‐specific immune complexes are present in chronic coronary heart disease. Methods and Results. The presence of Chlamydia‐specific circulating immune complexes was studied in 46 patients with chronic coronary heart disease and in control subjects. Chlamydial lipopolysaccharide‐containing immune complexes were detected with the antigen‐specific capture method, and they were present in 41% of patients and 15% of control subjects (p<0.01). The presence of C. pneumoniae antibodies in circulating immune complexes was studied by testing the specificity of antibodies derived from isolated and dissociated immune complexes by microimmunofluorescence testing and immunoblotting. The C. pneumoniae indexes based on the relative amount of immune complex‐derived antibodies and free antibodies were significantly higher among patients compared with control subjects (median, 1/8 versus 1/16;p<0.001). Immune complex bound antibodies showed specificity for 98‐kd and 42‐kd proteins of C. pneumoniae. Conclusions. The results suggest that the majority of the patients with chronic coronary heart disease have a chronic C. pneumoniae infection in which chlamydial components have an easy access to circulation to form immune complexes with preexisting antibodies. These findings give further evidence for the association of chronic C. pneumoniae infection with coronary heart disease. (Circulation 1993;87:1130‐1134)

The epidemiology and significance of Chlamydia pneumoniae

Chlamydia pneumoniae has recently been found to be a new chlamydial species transmitted directly from man to man, obviously through the respiratory tract. It is the commonest chlamydia of mankind but fortunately the overwhelming majority of infections are mild, it has been estimated that 10% of all pneumonias are caused by this species. Since the most important chlamydial infections are chronic in nature, the question of possible chronic C. pneumoniae infections is of paramount importance. Apart from being associated with chronic inflammatory processes of the respiratory system, a quite unexpected connection to coronary heart disease has now been suggested.

Aetiology of community-acquired pneumonia in children treated in hospital

Viral and bacterial antigen and antibody assays were prospectively applied to study the microbial actiology of community-acquired pneumonia in 195 hospitalised children during a surveillance period of 12 months. A viral infection alone was indicated in 37 (19%), a bacterial infection alone in 30 (15%) and a mixed viral-bacterial infection in 32 (16%) patients. Thus, 46% of the 69 patients with viral infection and 52% of the 62 patients with bacterial infection had a mixed viral and bacterial aetiology. Respiratory syncytial virus (RSV) was identified in 52 patients andStreptococcus pneumoniae in 41 patients. The next common agents in order were non-classifiedHaemophilus influenzae (17 cases), adenoviruses (10 cases) andChlamydia species (8 cases). The diagnosis of an RSV infection was based on detecting viral antigen in nasopharyngeal secretions in 79% of the cases. Pneumococcal infections were in most cases identified by antibody assays; in 39% they were indicated by demonstrating pneumococcal antigen in acute phase serum. An alveolar infiltrate was present in 53 (27%) and an interstitial infiltrate in 108 (55%) of the 195 patients. The remaining 34 patients had probable pneumonia. C-reactive protein (CRP), erythrocyte sedimentation rate and total white blood cell count were elevated in 25%, 40% and 36% of the patients, respectively, CRP was more often elevated in patients with bacterial infection alone than in those with viral or mixed viral-bacterial infections. No other correlation was seen between the radiological or laboratory findings and serologically identified viral, bacterial or mixed viralbacterial infections. By using a comprehensive serological panel, the causative agent could be found in over 50% of patients with pneumonia. We conclude that RSV and pneumococcus are the two most common organisms causing pneumonia in children. Our results suggest that mixed viral-bacterial aetiology is common in lower respiratory tract infections affecting children.

Circulating immune complexes containing chlamydial lipopolysaccharide in acute myocardial infarction

The presence of circulating immune complexes (IC) was studied using two detection methods specific for chlamydial lipopolysaccharide (LPS) in paired serum samples of 44 patients (30 men and 14 women) with acute myocardial infarction (AMI). Forty-four random controls were individually matched for locality, age and sex with the AMI patients. As specificity controls for the IC assays single serum samples from 29 patients with diseases characterized by the presence of circulating IC were used. Fifty-seven per cent of AMI patients, 12% of their random controls and 10% of the patient controls were shown to have chlamydial LPS-specific immune complexes in their sera (P less than 0.0001, AMI versus random and patient controls). This finding provides further evidence of the possible association of chronic chlamydial infection with AMI.

Evaluation of serological methods in the diagnosis of Chlamydia pneumoniae pneumonia during an epidemic in Finland.

A complement fixation (CF) test, a micro-immunofluorescence (micro-IF) test and an enzyme immunoassay (EIA) using Re-lipopolysaccharide as antigen were compared in the diagnosis of chlamydial infection in 136 mainly elderly patients hospitalized with community-acquired pneumonia during aChlamydia pneumoniae epidemic in Finland in 1986–1987. Chlamydial pneumonia was diagnosed in 58 (42.6 %) of the 136 pneumonia patients; 44 (75.9 %) of them could be shown by micro-IF to be caused byChlamydia pneumoniae, three byChlamydia psittaci and four byChlamydia spp. Only 5 (11.4 %) of 44 patients withChlamydia pneumoniae pneumonia were IgM-positive, indicating that the majority of cases were reinfections. In this population of mainly elderly patients the CF test was insensitive, being positive in only 6 (10.3 %) of 58 cases of chlamydial pneumonia. The EIA detected 72.4 % of cases and micro-IF 87.9 % of cases (including infections withChlamydia pneumoniae, Chlamydia psittaci andChlamydia spp.). In the EIA 77 % of positive cases were positive in serum samples taken a week apart, whereas the corresponding figure for micro-IF was 50 %. In micro-IF the measurement of IgA antibody levels is recommended and IgM-positive sera should be retested after removal of IgG antibody to avoid false-positive findings due to presence of rheumatoid factor. The collection of a third serum sample, for instance one month after onset, is also recommended, since half of the patients showed a diagnostic response in the micro-IF only in the sera taken one month apart.

The epidemic cycle of Chlamydia pneumoniae infection in eastern Finland, 1972–1987

The epidemic cycle of Chlamydia pneumoniae infection was examined in two areas in eastern Finland over a period of 15 years, 1972-87. The C. pneumoniae IgG antibody prevalence was determined with 5-year intervals in a random sample of the population aged 25-59 years. The total number of sera studied using immunofluorescence was 2387. In 1972 the antibody prevalence was 57% and it increased to 66% in 1977. Over the next 5 years the prevalence decreased to 44% in 1982, but by 1987 it had again increased to 59%. The temporal variation in prevalence was statistically significant (P < 0.001) and similar for both genders. Throughout the observation period the overall prevalence was 7-11% higher in men than in women (P < 0.01). The antibody prevalence increased with age, being the highest among the oldest study subjects of both genders. The periods of high and low prevalence alternated in an epidemic cycle (P < 0.001) of about 10 years.

Experimental Chlamydia pneumoniae infection in mice: Effect of reinfection and passive immunization

NIH/S mice were infected intranasally with Chlamydia pneumoniae isolate Kajaani 6 and rechallenged after either 28 or 70 days. A partial resistance to reinfection, indicated by a reduced recovery of live organisms, was noted at both time points of rechallenge: positive isolations from lung homogenates and/or bronchoalveolar lavage fluids were observed in fewer mice and the yields of isolated chlamydiae remained smaller, as compared to primary infection. However, a previous infection did not confer any protection against inflammatory changes. A strong peribronchial and perivascular inflammation with infiltrating lymphocytes and plasma cells was noted in the lungs of primary infected, as well as reinfected, mice. The effect of passive immunization was also studied. When mice were given convalescent or hyperimmune sera intraperitoneally before inoculation, lower C. pneumoniae isolation yields were detected. As in the rechallenge experiment, marked inflammation could still be seen in the lungs, now with polymorphonuclear leukocyte infiltration. The results suggest that immunological reactions play a role in the pathogenesis of C. pneumoniae infection. Antibodies may be important in reducing the amount of infective elementary bodies, but complete clearing of C. pneumoniae could not be achieved in these experiments, even less a protection against inflammatory lung changes.

Serological response to Chlamydia pneumoniae in patients with sarcoidosis

The antigen-specific serological response to Chlamydia pneumoniae was studied in 24 patients with sarcoidosis and compared to that seen in acute C. pneumoniae respiratory infection. By the micro-immunofluorescence test, five sarcoidosis patients had acute antibody, 15 had chronic antibody and four had no antibody against C. pneumoniae. By enzyme immunoassay, 20 sarcoidosis patients had antibody against ReLPS but that cross-reacts with chlamydial LPS. Immunoblot analysis of sera using purified C. pneumoniae elementary bodies showed that recognition of the 40 kDa C. pneumoniae major outer membrane protein was rare (20%). Reactivities with proteins with Mw of 42 K (70%), 60 K (65%), 98 K (55%) and 52 K (50%) were often noted. To study reactivity of chlamydial HSP 60 in sarcoidosis sera, sarkosyl-soluble (contains the 60 kDa HSP) and sarkosyl-insoluble (contains the 60 kDa structural protein) fractions of C. pneumoniae elementary bodies were prepared. The 60 kDa structural protein was recognized with equal frequency by sera from patients with sarcoidosis and acute respiratory infection, while the HSP 60 was more frequently recognized by sera with acute respiratory infection than sarcoidosis. Recombinant fusion proteins expressed from pGEX-2T containing overlapping DNA fragments of the C. pneumoniae 60 kDa HSP gene were purified. Different recognition patterns were identified for sera from sarcoidosis patients and from patients with acute C. pneumoniae respiratory infection.

The seroepidemiology of Chlamydiae in Finland over the period 1971 to 1987.

The seroepidemiology of chlamydial infections in the Finnish population was studied by analysing the prevalence of chlamydial complement fixing (CF) antibodies in patients sera sent for virus serological screening tests over 17 years from 1971 to 1987. The total number of sera studied was over 160,000. In the early 1970s, the prevalence of chlamydial CF antibodies (CF titres greater than or equal to 8) was low (less than 2%), but later the proportion of seropositive cases rose, and in 1976, 18% of sera contained antibodies. In 1984, the seropositivity rate was over 31%. The prevalence of high chlamydial CF titres (titres greater than or equal to 64) also showed annual variation. In general, under 1% of sera contained chlamydial CF antibodies in high titre, but in 1979 and 1984, distinct peaks occurred when 1.3% and 1.4% of sera, respectively, had titres greater than or equal to 64. The age-related antibody positivity rate showed a decline during early infancy, an increase in childhood and adolescence, and a stable level in adulthood when approximately 20% of the sera contained antibodies. The chlamydial antigen used in this survey was genus-specific, i.e. it detects antibodies against all chlamydial species. Epidemiological data support the hypothesis that infections due to a novel chlamydial species, TWAR chlamydia, are the most likely explanation for the relatively frequent occurrence of chlamydial CF antibodies and for the variation in CF antibody prevalence.