Clinical Microbiology Reviews, January 2002, p. 1-20, Vol. 15, No. 1
0893-8512/01/$04.00+0 DOI: 10.1128/CMR.15.1.1-20.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Chlamydia pneumoniae and Atherosclerosis: Critical Assessment of Diagnostic Methods and Relevance to Treatment Studies
Jens Boman1 and Margaret R. Hammerschlag2*Department of Virology, Umeå University, Umeå, Sweden,1 Division of Pediatric Infectious Diseases, Department of Pediatrics, State University of New York Downstate Medical Center, Brooklyn, New York2
ASSOCIATION OF CHLAMYDIA PNEUMONIAE AND CARDIOVASCULAR DISEASE Role of Inflammation and Infection in Cardiovascular Disease ASSOCIATION OF C. PNEUMONIAE AND CARDIOVASCULAR DISEASE: BACKGROUND ROLE OF SEROLOGIC TESTING IN DIAGNOSIS OF C. PNEUMONIAE INFECTION MIF Assay Problems with Standardization of the MIF Assay Enzyme Immununoassays Serologic Diagnosis of Chronic C. pneumoniae Infection Current Recommendations for C. pneumoniae Serologic Testing SEROEPIDEMIOLOGIC STUDIES IDENTIFICATION OF C. PNEUMONIAE IN VASCULAR TISSUE Early Detection Studies Subsequent Detection Studies Immunohistochemical Detection Methods Cell Culture PCR Correlation of the Presence of C. pneumoniae with the Degree of Atherosclerosis Alternative Types of Specimens Correlation of Serology with Identification of C. pneumoniae in Vascular Tissue Conclusion: Detection of C. pneumoniae in Vascular Tissue CLINICAL TREATMENT INTERVENTION STUDIES Treatment of C. pneumoniae Infection: Background Retrospective Case-Control Studies Prospective Controlled Treatment Trials Effect of Treatment of C. pneumoniae in Animal Models of Atherosclerosis CONCLUSIONS REFERENCES
SUMMARY
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A number of studies have found that inflammation of the vessel wall plays an essential role in both the initiation and progression of atherosclerosis and erosion and fissure and the eventual rupture of plaques. Chlamydia pneumoniae is one of the infectious agents that have been investigated as possible causes of this inflammation. Initial studies of the association of C. pneumoniae and cardiovascular disease (CVD) were seroepidemiologic, and these were followed by studies in which the organism was identified in vascular tissue from patients with CVD by electron microscopy, PCR and immunocytochemical staining (ICC). C. pneumoniae has also been isolated by culture from vascular tissue in a small number patients. However, no single serologic, PCR, or ICC assay has been used consistently across all studies. The assays used are also not standardized. Recent studies of serologic and PCR assays for diagnosis of C. pneumoniae infection have suggested that there may be substantial interlaboratory variation in the performance of these tests. It now appears that some of the inconsistency of results from study to study may be due, in part, to lack of standardized methods. Although initial seroepi-demiologic studies demonstrated a significantly increased risk of adverse cardiac outcome in patients who were seropositive, subsequent prospective studies found either small or no in-creased risk. In addition to the lack of consistent serologic criteria, recent evaluations have demonstrated inherent problems with performance of the most widely used serologic methods. Most importantly, we do not have a reliable serologic marker for chronic or persistent C. pneumoniae infection.
ASSOCIATION OF CHLAMYDIA PNEUMONIAE AND CARDIOVASCULAR DISEASE
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Atherosclerotic heart disease is the leading cause of morbidity in the Western Hemisphere, including the United States (37). The clinical manifestations of atherosclerosis range from coronary artery disease (CAD) to cerebrovascular disease. Conventional risk factors including cigarette smoking, hypertension, and high serum lipid levels do not fully explain the incidence, prevalence, and distribution of CVD. A number of studies have found that inflammation of the vessel wall plays an essential role in both the initiation and progression of atherosclerosis, erosion, and fissure and the eventual rupture of plaques (69, 70, 85, 88, 95, 116). The hypothesis that inflammation contributes to the pathogenesis of atherosclerosis is over 100 years old, dating back to Virchow in 1859 (140a) and Osler in 1908 (102a). Recent studies have demonstrated that various markers of systemic inflammation can predict future cardiovascular events including nonfatal and fatal myocardial infarction and stroke (2, 70, 115, 116). Acute-phase reactants such as C-reactive protein (CRP) and fibrinogen have been noted to be elevated in patients with unstable angina (88, 116). A meta-analysis of 14 studies found a significant association of elevated levels of CRP and fibrinogen with CAD (24). Therapy with aspirin, in addition to affecting platelet function, reduces inflammation as measured by levels of CRP and may prevent myocardial infarction by this mechanism (115). However, only subjects in the highest quartile showed any benefit of aspirin use; 75% of men showed marginal or no benefit.
Although inflammation is present, the exact cause of this inflammation in CVD is still not known. CRP is a nonspecific marker of various stimuli including tissue damage, smoking, and infection.
Infectious agents that have been investigated as possible stimuli include viruses, specifically cytomegalovirus, human herpesviruses, and enteroviruses, and bacteria including Helicobacter pylori, the agents of periodontal disease, and Chlamydia pneumoniae (6, 20–22, 31, 33, 88, 98, 103, 118, 122, 131).
ASSOCIATION OF C. PNEUMONIAE AND CARDIOVASCULAR DISEASE: BACKGROUND
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C. pneumoniae was first characterized in 1986 and is now recognized as a common cause of community-acquired respiratory infection in adults and children (11, 42). The first report of a possible connection between C. pneumoniae and atherosclerosis came from a serologic study performed in Finland in 1988 (124). Using the microimmunofluorescence (MIF) method, Saikku et al. found that patients with proven CAD were significantly more likely to have anti-C. pneumoniae antibodies than were control patients selected at random. Since this initial report, almost 500 papers have been published on the association of C. pneumoniae and atherosclerosis; almost 300 were published in 2000. The bibliography includes a large number of reviews of the subject, some quite extensive (16, 21, 29, 37, 69, 98). Seroepidemiologic studies were followed by studies in which the organism was identified in vascular tissue from patients with CVD by electron microscopy, PCR, and immunocytochemical staining (ICC). C. pneumoniae has also been isolated by culture from vascular tissue in a small number of studies (4, 59, 83, 113). Animal studies, including mouse and rabbit studies, have demonstrated that C. pneumoniae disseminates systemically after respiratory infection and also appears to either induce or enhance the development of atherosclerosis (35, 95, 123), although these results have not been consistent (145). A recent paper by Wright et al. (150) has demonstrated that infectious agents are not necessary for initiating murine atherosclerosis. These studies have led to primary- and secondary-intervention studies using antibiotics directed at a putative intravascular C. pneumoniae infection.
However, no single serologic, PCR, or ICC assay has been used consistently across all studies. The assays used are also not standardized. Recent studies of serologic and PCR assays for diagnosis of C. pneumoniae infection have suggested that there may be substantial interlaboratory variation in the performance of these tests (5, 108, 109, 113). This could lead to sizable random measurement errors resulting in underestimation of the effect due to regression-dilution bias; alternatively, systematic measurement biases, including lack of blinding of disease status, could lead to overestimation of risk. The issue of assay performance has rarely been addressed in most studies. The major purpose of this review is to examine the impact of methods on studies of the association of C. pneumoniae and CVD and the relevance to treatment studies.
ROLE OF SEROLOGIC TESTING IN DIAGNOSIS OF C. PNEUMONIAE INFECTION
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Although the potential difficulties in performance of the MIF assay, including the subjective component in reading the assay results, have been mentioned numerous times (21, 23, 37), the extent of laboratory-to-laboratory variation and interpretation of the results was not specifically addressed. Similarly, the other serologic tests used such as enzyme immunoassay (EIA) and time-resolved fluorimetry (TRF) included nonstandardized in-house assays and commercially available kits. However, none of these assays are either cleared or approved by the Food and Drug Administration for the diagnosis of C. pneumoniae infection. One of the major issues not addressed in any of the seroepidemiologic studies is the correlation of serology with infection as defined either by isolation of the organism in culture or by PCR. Does an immunoglobulin G (IgG) or IgA titer of 64 really mean that a person has a chronic infection with C. pneumoniae?
Initially, as isolation of C. pneumoniae was felt to be difficult, emphasis was placed on serologic diagnosis using the MIF assay. The MIF assay was first developed as a seropidemiologic tool for C. trachomatis infection (142). Grayston et al. (42) proposed a set of criteria for serologic diagnosis of C. pneumoniae infection by a modification of the MIF test, specifically using C. pneumoniae TW183 elementary bodies instead of the 15 C. trachomatis type strains. Acute infection was defined as a fourfold rise in IgG titer, a single IgM titer of
16, or a single IgG titer of 512. Past or preexisting infection was defined on the basis of an IgG titer between 16 and 512. It was further proposed that the pattern of antibody response in primary infection differed from that seen in reinfection. In initial infection, the IgM response appears about 3 weeks after the onset of illness and the IgG response appears at 6 to 8 weeks. In reinfection, the IgM response may be absent and the IgG occurs earlier, within 1 to 2 weeks. Because of the relatively long period until the development of a serologic response with the MIF test in primary infection, the antibody response may be missed if convalescent-phase sera are obtained too soon, i.e., earlier than 3 weeks after the onset of illness. The criteria for use of a single serum sample have not been correlated with the results of culture and are based mainly on data from adults. Acute, culture-documented infection can occur without seroconversion, especially in children (11, 56, 76). However, culture-positive, MIF-negative children do develop an immune response to C. pneumoniae as measured by immunoblot analysis (76). The correlation between culture and serology is higher in adults, but culture-documented infection in adults with pneumonia can also occur in the absence of seroconversion or detectable antibody by MIF (52).
Background rates of seropositivity by MIF can also be very high in some adult populations, often exceeding 80% (57, 68). Hyman et al. (57), as part of a study of asymptomatic C. pneumoniae infection among subjectively healthy adults in Brooklyn, N.Y., also found that 17% of these healthy culture- and PCR-negative individuals had evidence of "acute infection" (IgG of 512 and/or IgM of 16). Therefore, one cannot really establish or rule out a diagnosis of C. pneumoniae infection on the basis of a single acute-phase serum titer by MIF. Evidence also suggests that antibody titers (IgG and IgM) may fluctuate significantly over time. Dean et al. (26) described two patients with persistent C. pneumoniae respiratory infection, documented by culture, who were infected for up to 7 years. During this period, IgM and/or IgG MIF serology was diagnostic for infection at only 25 to 50% of visits when the patients were symptomatic and culture positive.
Results of immunoblotting studies have also suggested that there may be antigenic diversity between the isolates of C. pneumoniae that may result in different results if different antigens are used in the MIF assay (76, 140). Some IgG antibody may result from a heterotypic response to other chlamydial species since there are cross-reactions with the major outer membrane protein (MOMP) between the four species as well as cross-reactions due to the genus lipopolysaccharide (LPS) antigen (18, 106). Other organisms that have been reported as possibly causing cross-reactions in the MIF assay are Bartonella and Bordetella pertussis (62, 87). The latter species could be significant since adults with pertussis frequently present with a chronic cough or severe bronchitis, which is a clinical presentation often ascribed to C. pneumoniae. Recent studies have found significant homology between human and C. pneumoniae HSP60 and Escherichia coli GroEL (101). Harkonen et al. (55) also reported recently that picornavirus proteins share antigenic determinants with HSP60/65, including the HSP60 proteins of humans and C. pneumoniae, which conceivably could also lead to cross-reactions in the MIF assay.
As discussed above, the MIF test is not standardized and many laboratories use in-house assays. Reading the slides has a very large subjective component and requires a very experienced microscopist. A recent study by Peeling et al. (108) attempted to address the problem of interlaboratory variation in the performance of the MIF test by sending a panel of 22 acute- and convalescent-phase sera to 14 different laboratories. Nine of the laboratories used an in-house MIF. The isolates used in the majority of the assays were either AR-39 or TW-183. Three laboratories used one of two commercially available kits, which used either AR-39 or Kajaani 6 as the test isolate. The remaining two laboratories used the Washington Research Foundation kit, which uses TW-183. The overall agreement (to get within one twofold dilution of the "gold standard" as read at the University of Washington) of all laboratories was 80%. The range was 50 to 100% depending on the isotype. Agreement for serodiagnostic criteria were 69% for negative, 68% for "chronic," and 87% for a fourfold increase of IgG titer.
Since it was not clear if this poor reproducibility was secondary to individual subjective differences in reading the slides or to the use of different assays with different antigens, Peeling et al. (109) performed another study comparing the University of Washington assay, which uses TW183, and a commercially available kit which uses AR-39 as the antigen (MRL Diagnostics, Cypress, Calif.). A panel of 10 sera was sent to 17 laboratories in the United States and Europe. Using the same criteria for agreement as the previous study, agreement for IgM titers in individual serum samples, using the University of Washington kit, ranged from 53 to 100% and agreement for IgG titers ranged from 47 to 100%. There was 100% agreement for IgG titers in only 3 of the 10 sera. Compared to the reference standard at the University of Washington, the agreement for the remaining seven sera ranged from 47 to 81%. The titers in the discrepant samples reported by the other laboratories tended to be lower than the reference standard. The results were similar for the MRL kit; the agreement ranged from 80 to 100% for IgM and 60 to 100% for IgG, but there was 100% agreement for IgG titers in only 2 of the 10 sera.
Although EIAs offer the promise of standardized performance and objective end points, none have been adequately evaluated compared to culture or PCR (27, 75, 110, 111, 140). Most have been compared only to MIF. None have Food and Drug Administration clearance or approval for use in the United States. One commercial assay, the Medac rELISA, uses a recombinant LPS antigen; others are based on LPS-extracted elementary bodies or synthetic peptides (140). EIAs based on LPS are genus specific and thus may have a higher risk for cross-reactions not only with other Chlamydia species but also with other genera of bacteria. These kits can measure IgG, IgM, and IgA antibodies, but the cutoffs vary from kit to kit and the criteria for a positive result (acute infection, past infection) can be very complex (75, 110, 111, 140). Kutlin et al. (75), in a study from the United States, compared the recombinant LPS EIA to culture and found sensitivities ranging from 13% for IgM antibody in children to 78% for either IgA or IgG antibody in adults with respiratory infection. Specificities compared to culture ranged from 21 to 91%. Recently Persson and Haidl (110) reported cross-reactions with the rEIA and parvovirus, primarily for IgM, but it was also seen to a lesser extent with IgG and IgA. This cross-reaction was not seen in MIF.
Anti-C. pneumoniae IgA antibody has been promoted as a marker of chronic C. pneumoniae infection. The rationale for this is that as the biological half-life of serum IgA is less than 7 days, compared to 23 days for IgG, the presence of this antibody implies an active or persistent infection. Determination of IgA antibody by MIF is also dependent on the IgA conjugate used. The presence of rheumatoid factor and high levels of IgG can also interfere with determination of the IgA titers (64a, 140a). Wang and Grayston (143) examined sera from young adults with acute respiratory infections and older adults with chronic obstructive pulmonary disease and found that only 17 to 42% of those with IgG and/or IgM antibody titers suggestive of acute or recent infection by MIF had detectable anti-C. pneumoniae IgA.
As part of a recent workshop on standardization of C. pneumoniae diagnostic methods, the Centers for Disease Control and Prevention (27) proposed some modifications of the serologic criteria for diagnosis. Although the MIF test was considered to be the only currently acceptable serologic test, the criteria were made significantly more stringent. Acute infection, using the MIF test, was defined by a fourfold rise in the IgG titer or an IgM titer of
16; use of a single elevated IgG titer was discouraged. An IgG titer of 16 was felt to indicate past exposure, but neither elevated IgA titers nor any other serologic markers were felt to be validated indicators of persistent or chronic infection. Available data support these recommendations, especially the poor predictive value of a single high IgG titer (57, 68).
SEROEPIDEMIOLOGIC STUDIES
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The original investigations of the association of C. pneumoniae and CVD were based on serology, predominantly using the MIF test. Saikku et al. (124) reported an association of anti-C. pneumoniae titers, specifically an IgG titer of >128 and/or an IgA titer of >32, with acute myocardial infarction (MI) and chronic CAD in a retrospective study of men enrolled in the Helsinki Heart Study. A subsequent, larger study from the same group also found an association of chronic C. pneumoniae infection, as defined by the above serologic criteria, with the risk of developing CAD; the odds ratios (OR) were 2.3 (95% confidence interval [CI], 0.9 to 6.2) for elevated IgA titers (125). These studies were followed by an additional 16 seroepidemiologic studies of the association of C. pneumoniae and CAD and cerebrovascular disease published through 1997, involving some 2,700 patients (21). These studies, and the topic of infection and CVD in general, were subjected to a detailed review and statistical analysis by Danesh et al. in 1997 (21). The majority of these studies were consistent in finding an association of C. pneumoniae antibodies and CVD, with ORs ranging from 1.2 to greater than 8. In some, there was also an association of increasing ORs with increasing antibody titers. Only one of these studies failed to find an association of seropositivity to C. pneumoniae with an increased risk of CVD (144). However, these studies varied greatly in terms of population, criteria for defining CVD, and adjustment for possible confounding variables such as age and smoking, which may make them prone to bias. Only 3 of these studies were prospective; 15 were cross-sectional with population or other controls. Most of these studies used MIF as the serology test. Only four of the studies specified that the microscopist performing the MIF was blinded to the clinical status of the patients. Most importantly, there was little consistency from study to study in the serologic criteria used to define past, chronic, or persistent infection by the MIF test. Various combinations of IgG and IgA and different antibody titer cutoffs were used. In some cases the titers selected were determined post hoc after the data had been analyzed as those that had the greatest association, and in some cases the same investigators used different serologic criteria in different studies (24, 92, 125, 136, 137). In most studies, sera were also obtained only once, which does not take into account the possibility that antibody titers may fluctuate over time (26).
Danesh et al. (23) published the results of a large population-based prospective study of the association of anti-C. pneumoniae IgG titers and CAD with another meta-analysis of 14 other prospective studies published through May 2000. Three of these studies were included in the original metanalysis published in 1997 (21, 44, 93, 125). These studies now included a total of 3,169 cases of nonfatal myocardial infarction or death from CAD. All studies adjusted for smoking and other classic risk factors for CAD. In contrast to the earlier series and meta-analysis, these recent prospective follow-up studies have not generally supported the association of C. pneumoniae infection, as defined by the presence of IgG and/or IgA antibody, and CVD. Danesh et al. (23) found a combined OR of 15 studies of 1.15 (95% CI, 0.97 to 1.36) for IgG antibody. In nine studies that reported risk in association with IgA titers, the combined OR was 1.13 (95% CI, 0.90 to 1.41) (23, 41, 99, 125, 132, 141).
There was a great deal of heterogeneity among these studies in terms of the serologic tests and criteria used for seropositivity. In some studies, an IgG and/or IgA titer of 64 was used as an indicator of chronic infection; in others, the same or similar criteria were used as indicators of past infection (Table 1). The serologic tests and criteria used in 12 of the prospective studies analyzed by Danesh et al. (23) and 2 subsequently published studies (1, 20) are shown in Table 1. Three of the prospective studies analyzed by Danesh et al. (23) were available only as abstracts or as a Letter to the Editor; therefore, details about the serologic methods used were limited. These are not included in the table. Nine studies used MIF assays, and all were in-house tests (20, 44, 93, 99, 117, 118, 125, 131, 132). One study used a commercial whole-cell indirect immunofluorescence assay (1). The antigen used was specified in only four of the MIF assays, two used TW-183 and one each used Kajaani 6 and IOL-207. Two studies used EIAs, and one used two different in-house-developed assays, one genus specific and one purportedly species specific. TW-183 was the antigen, and the elementary bodies were treated with sodium periodate to remove the genus-specific LPS antigen (103). The other study used a commercially available EIA, Medac rELISA, which uses a recombinant Chlamydia genus-specific LPS antigen (41). Two studies (23, 141), including the Danesh et al. (23), used TRF, which was previously reported to correlate well with MIF (149), but the antigen and criteria used were not specified. The IgG titers used as the cutoff for seropositivity in the studies that used MIF assays ranged from 8 to 32. Three studies defined an anti-C. pneumoniae IgG titer of 64 as indicating past or chronic infection, or as being "elevated"; one study used an IgG titer of 128; and one study did not specify a titer. In the studies that used an EIA or TRF, the titers were frequently not specified. The antibody levels in EIAs were expressed as optical density rather than titer, which makes comparisons to the results of the MIF studies difficult.
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TABLE 1. Diagnostic criteria for C. pneumoniae infection used in prospective seroepidemiologic studies
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IDENTIFICATION OF C. PNEUMONIAE IN VASCULAR TISSUE
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The first study suggesting the presence of C. pneumoniae in atherosclerotic plaques of coronary arteries was performed by Shor et al. in South Africa (128). The study was done during autopsy of young adult men 8 to 36 h after death and on hearts received for examination at the Pathology Department. One thousand coronary arteries were examined, and 10 with early lesions were selected for electron microscopy (EM). All samples showed pear-shaped structures. Seven atheroma lesions were examined both by EM and by Chlamydia immunoperoxidase staining with Chlamydia genus-specific (CF-2), C. pneumoniae-specific (RR-402), and C. trachomatis-specific (KK-12) monoclonal antibodies. By EM, all seven cases showed vesicles with similar C. pneumoniae-like organisms in the lipid-rich core of the fibolipid plaque. Five (71%) of seven EM-positive cases were positive by immunoperoxidase staining with Chlamydia genus-specific and C. pneumoniae species-specific antibodies but negative for the C. trachomatis species. Coronary artery tissues from five control cases were negative by immunoperoxidase staining.
The specimens examined were selected from over 1,000 autopsies done over a 2-year period. Consequently, the finding should not be taken to indicate a strong association of C. pneumoniae with coronary artery atherosclerosis. However, the study was not designed to provide population-based data but, rather, to find evidence that the organism can be detected in atherosclerotic lesions. In a subsequent study, Kuo et al. (71) reported further data on the possible physical association of C. pneumoniae with coronary atherosclerosis in additional South African autopsy cases. In that study, ICC, PCR, EM, and isolation attempts were performed (Table 2). A total of 16 early lesions or fatty streaks and 20 later lesions or fibrolipid plaques of coronary arteries from 31 men and 5 women were studied. For the ICC, a Chlamydia genus-specific monoclonal antibody (CF-2) was used for initial screening of specimens and screening-positive samples were sectioned for staining using a C. pneumoniae-specific monoclonal antibody (RR-402). PCR was used with C. pneumoniae-specific primers. By ICC, 15 (42%) of 36 tested cases were positive; corresponding figures for the PCR were 13 (43%) of 30 analyzed samples. Of the 30 samples tested by ICC and PCR, only 8 (27%) were positive by both methods, 8 (27%) were positive by one method only (5 by PCR and 3 by ICC), and 14 (47%) were negative in both assays. EM revealed Chlamydia-like elementary bodies in 6 (29%) of 21 atheromatous plaques, of which 2 were positive by both ICC and PCR, 1 was positive by ICC only, 1 was positive by PCR only, and 2 were negative by both ICC and PCR. All attempts to isolate Chlamydia organisms were negative. Two cases were selected for sequence analysis of PCR-amplified products: the DNA sequence was identical to the C. pneumoniae sequence. The agreement between the methods was not very high, since only 2 cases (10%) out of 21 positive by either method were positive by all three detection methods (ICC, PCR, and EM) used (Table 2). Discrepancies may arise from testing different areas of the lesions and/or poor sensitivities, specificities, or both for the methods used in the study. Cell culture was not able to support the presence of C. pneumoniae in the plaques in any case, but the conditions were not optimal since postmortem samples obtained many hours to days after death was used. C. pneumoniae was detected in the plaques of six (75%) of the eight cases that were negative by serology. This is somewhat paradoxical since the original hypothesis that C. pneumoniae and atherosclerotic coronary heart disease (CHD) were related was based on increased seroprevalence in patients with CHD compared with controls (124). However, the serologic conditions may have been suboptimal since hemolysed postmortem sera were used.
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TABLE 2. Detection of C. pneumoniae in atheromatous tissue specimens
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After these initial studies on the detection of C. pneumoniae in vascular tissue, a large number of studies dealing with C. pneumoniae and atherosclerotic disease were performed to confirm or refute these early observation (3, 4, 6, 7–9, 19, 25, 30, 32, 33, 35, 38, 59, 64–66, 74, 80, 82–85, 94, 97, 100, 104, 105, 107, 112, 113, 129, 133, 138, 147, 151). When different methods, including PCR, ICC, culture, and EM, were used, the reported prevalence of C. pneumoniae in atheromatous tissue varied substantially (Table 2), and we now span the spectrum from research groups who claim to detect C. pneumoniae in vascular tissue commonly and easily to groups who cannot detect the bacterium in atherosclerotic tissue under any circumstances. It is recommended that persons processing the specimens and interpreting the results for research studies be blinded to the patient status and results of other testing (27). Unfortunately, most reports do not specify how blinding was ensured, which is of major concern. Some reports, however, include a careful description of how the blinding was performed (129). This section will focus on these detection studies, and especially on the methods used and potential methodological limitations in the studies. Between 1992 and 2000, more than 40 studies were published on the detection of C. pneumoniae in vascular tissue. The results of 43 studies are summarized in Table 2; these studies include 2,644 atherosclerotic specimens from various human arteries and some veins. The most commonly used techniques are ICC and PCR; other techniques used are cell culture, EM, immunofluorescence (IF), and in situ hybridization (ISH). By immunostaining techniques using a Chlamydia genus-specific antibody, 336 (49.7%) of 676 examined specimens were positive; with a C. pneumoniae-specific antibody, 202 (45.6%) of 443 examined specimens were reported as positive. Corresponding figures are 558 (24.3%) of 2,294 for PCR, 33 (7.3%) of 451 for culture and 38 (39.2%) of 97 analyzed specimens for EM. In most reports, all positive results obtained by either of the methods have been accepted as true-positive findings, despite often substantial disagreement and lack of concordance (Table 2). Using this approach, the prevalence of C. pneumoniae in atheromatous tissue may be very high. However, the interpretation of the discrepant results obtained by similar and different diagnostic methods is problematic in the absence of a "gold standard" and is of major concern. If a more reliable approach is used as a gold standard to define a positive specimen, i.e., a specimen (e.g., an atherosclerotic plaque) is accepted as true positive if it is positive by two (or more) independent C. pneumoniae-specific techniques, the prevalence of C. pneumoniae in atheromatous tissue will drop significantly to 84 (3.14%) of 2,679 atheromatous specimens. However, only 502 samples (18.74%) were analyzed by two (or more) independent C. pneumoniae-specific techniques (Table 2). If only these studies are included in the calculation, the prevalence is 16.73% for atheromatous tissue specimens. Since some of the studies used only ICC with a species-specific antibody on screening-positive samples (screening with genus-specific antibody), this calculation is biased, resulting in a very high prevalence of C. pneumoniae-positive specimens. If such studies are excluded from the calculation, the prevalence of true-positives specimens is 76 (15.14%) of 502, a figure very close to that obtained by cell culture (16%) in studies performed with several passages (5, 78). The total number of control specimens (n = 637) included in the detection studies on vascular tissue is much smaller (24%), and several reports have not included control tissue at all (Table 3). In studies that examined control "normal" tissue, the reported prevalence of C. pneumoniae has been low (Table 3), with 10 positives (3.21%) out of 312 samples for ICC using a Chlamydia genus-specific test, 12 (13.33%) out of 90 using a C. pneumoniae species-specific ICC test, and 25 (4.56%) out of 548 for PCR. None of 22 examined by culture, none of 3 examined by EM, and none of 2 analyzed by ISH were positive. The choice of controls has been criticized because of inadequate numbers and inadequate age matching (134). Thus, a lower prevalence of C. pneumoniae in control tissue may simply be due to the use of younger persons in the control group, i.e., persons with a lower prevalence of vascular C. pneumoniae infection (73). When choosing a control population, it is also important to take into account the geographical location of the subjects as well as the time (month and year) of the samples collected since infection with C. pneumoniae has been documented to occur in local epidemics.
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TABLE 3. Detection of C. pneumoniae in normal tissue specimens
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Antigen detection methods with the use of genus- or species-specific antibodies labeled with a peroxidase (immunocytochemistry and immunohistochemistry) or a fluorescent (immunofluorescence) marker are commonly used to identify Chlamydia in vascular tissue (Table 4). These techniques may all allow localization of the organisms in specific cells and areas. However, most of these techniques require a subjective reading. Considerable difficulty has been experienced with the immunofluorescence technique because of nonspecific background staining and the morphological heterogeneity of C. pneumoniae elementary bodies (102, 138). Further, background staining made the interpretation of the results of ICC difficult for 28 (80%) of 35 specimens tested by Shor et al. (129), and great difficulty was experienced with the ICC test by Ong et al. (102) because of nonspecific staining. Of major concern is that these techniques may yield results of decreased specificity when applied to atheromatous tissue (146). This may be due to the nonspecific staining of inflammatory cells present in inflamed atheromatous vascular tissue (134). Consequently, it is important to be certain that the antigenic components identified by this technique are C. pneumoniae specific and not due to a change of normal tissue antigenicity in atherosclerotic tissue. The variation of type, specificity, and avidity of different monoclonal antibodies may lead to false-positive and false-negative results (27). Detection rates vary widely (from 14 to 100%), and there is often poor correlation between antigen detection methods and DNA detection methods in the same atherosclerotic plaque (Table 2). It is possible that some of these discrepancies are due to false-positive results in the ICC test because of lack of an accepted and standardized approach to the method and interpretation of results (27). Ericson et al. (30) used two different immunological tests for detecting C. pneumoniae in atherosclerotic tissue: direct immunofluorescence and immunoperoxidase stain. While both tests were positive in a large proportion of specimens from patients with severe atherosclerosis (36 [86%] of 42 and 32 [80%] of 40, respectively), the agreement was low for specimens from patients with mild atherosclerosis (1 [6%] of 18 and 6 [38%] of 16, respectively). Consequently, it is important to be certain that these antigens are Chlamydia specific and not damaged atherosclerotic tissue that may have higher affinity for these antibodies than does normal vascular tissue (146). To obtain results as reliable as possible, guidelines have recently been formulated (27). For the choice of antibodies, it is recommended that two Chlamydia antibodies and two negative control antibodies always be used. It is also recommended that control tissue (positive and negative) be carefully selected and used consistently with each experiment and incubated with all four antibodies. Human and chlamydial heat shock proteins have highly conserved and homologous antigen sites (101). As a consequence, such antigens may interfere in tissue diagnostics, resulting in cross-reactions and false-positive results. Since only a very small portion of the lesion are generally examined by these in situ techniques, a negative result does not exclude the presence of C. pneumoniae in the plaque.
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TABLE 4. Description of methods for antigen detection of C. pneumoniae in vascular tissue
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Culture of the organism in cell culture is the prevailing gold standard used to demonstrate current infection by C. pneumoniae and to establish viability and thus infectivity. However, the use of culture for the detection of C. pneumoniae is problematic because of the difficulty in growing C. pneumoniae in cell culture, especially from tissue samples (27). As a result, culture results generated by different laboratories are not always reproducible. Some investigators report regular recovery of viable C. pneumoniae (6, 83), whereas other groups have reported low isolation rates (71, 74, 144).
The first study providing evidence for the presence of viable C. pneumoniae in the atheroma of a patient with severe coronary atherosclerosis was published 1996 (113); a second isolate from atheromatous tissue was recovered from a carotid artery (59). A highly sensitive culture system was developed by Maass et al. (83), who examined 70 coronary artery specimens for the presence of viable C. pneumoniae bacteria and for C. pneumoniae DNA. Atherosclerotic lesions from 53 coronary endarterectomy and 17 restenotic bypass specimens were cultured and subjected to nested PCR. Three segments per coronary sample were used; each plaque suspension was divided for PCR and cell culture in HEp-2 cells. The cell culture was performed with at least 10 serial passages and stained with a fluorescein isothiocyanate-conjugated C. pneumoniae-specific monoclonal antibody. For confirmation and enhancement of sensitivity of the nested PCR, nonradioactive DNA hybridization was performed. For control purposes, 7 arteries and 10 segments from the aortic wall, without macroscopic evidence of atherosclerosis, were available for PCR testing. Replicating C. pneumoniae isolates were recovered by cell culture from the occluded coronary arteries of 11 (16%) of 70 patients; 6 (54%) of the 11 isolates could be permanently propagated by serial subcultures. The isolates were identified in passages 2 to 5. Isolation was successful in all three arterial segments examined for six patients, in two segments for three patients, and in one segment only for two patients. The nested PCR yielded positive results with the arterial samples of 21 (30%) of 70 patients, including all culture-positive patients. Nine (82%) of the patients with a parallel positive culture result had positive PCR results in all three separately examined coronary artery segments, and two (18%) were positive in two of three segments. Of the 10 culture-negative, PCR-positive patients, 7 (70%) were positive for C. pneumoniae DNA in three segments and 3 (30%) were positive in two segments. All 17 control samples without macroscopically evident atherosclerosis were PCR negative. DNA sequence analysis of the PCR products of two continuously propagated coronary isolates revealed 100% sequence identity to a C. pneumoniae reference isolate. Thus, these cardiovascular strains do not appear to be different from respiratory tract isolates.
In this study, it was shown that it is possible to isolate viable C. pneumoniae from atherosclerotic coronary artery specimens if a sensitive culture system with several passages and serum-free medium are used. All isolates except two were confirmed by culture of a parallel segment of the same artery. Further, coronary artery specimens from all isolation-positive patients were PCR positive. None of the isolation- or PCR-positive patients was seronegative for C. pneumoniae in a C. pneumoniae-specific MIF test. Contamination is very unlikely since the PCR and culture results were in agreement and all 17 negative controls were negative. The results were confirmed in another study by the same group (85) and in a study by another group (4). Since increased number of passages may result in a concentration of cell debris resulting in nonspecific staining (27) and since the risk of contamination increases with increased number of passages, it is important to include several negative controls on each plate. It is important to note that true-positive C. pneumoniae cultures produce inclusions, not just elementary bodies, and can be passaged and detected by PCR.
The expansion of C. pneumoniae from a respiratory pathogen to its association with chronic diseases including asthma (48, 49) and atherosclerosis (124, 128) and the shortcomings of classical diagnostic methods have created a requirement for sensitive methods to detect this organism. Nucleic acid amplification technology was employed to meet this need. The ability of the PCR method to rapidly amplify small amounts of specific DNA or RNA, often in a background mixture of nucleic acids from the host and from other microorganisms, has made this technique a potentially valuable diagnostic tool. By PCR it may be possible to detect DNA and RNA of microorganisms that are present in small numbers, nonviable or growing slowly, or present in material not suitable for culture. However, several problems remain, mostly involving sensitivity and specificity issues and validation of new assays (14). Important issues include the choice of appropriate primers, optimization of the assay, extraction of nucleic acids, detection of amplification products, and prevention and identification of false-positive and false-negative results (14). Therefore, before nucleic acid amplification techniques can be widely adopted for the detection of C. pneumoniae, these problems must be solved. Since a variety of different PCR-based assays have been developed, another important issue is the comparison of these methods and the standardization of protocols. At present the lack of a consensus standard makes it difficult to evaluate methods, and, thus far, few PCR tests for detecting C. pneumoniae DNA have been sufficiently evaluated (27). The PCR technique is commonly used, alone or in combination with other diagnostic methods (Tables 2 and 5), for the detection of C. pneumoniae in the cardiovascular system. C. pneumoniae DNA has been detected in atherosclerotic plaques in coronary arteries (17, 83, 128), carotid arteries (19, 43, 59, 82), arteries of the lower extremities (74), abdominal aortic aneurysms (65, 102, 112), sclerotic heart valves (66, 100), and pulmonary arteries (129) and in atrial tissue from heart transplant donors (133). However, the frequency of positive results varies considerably (from 0 to 100%) among the studies (3, 65, 80); the main reason for this variability may be the use of different DNA extraction procedures and PCR protocols. Consequently, even if samples from the same patients are investigated by different laboratories, the variation of positive findings may be significant (5, 113). The most important steps in identifying false-positive results include the use of several negative tissue controls in each run. These controls should be processed in parallel with the clinical specimens throughout the DNA extraction and detection of amplification product. To further increase the specificity, one may need to reextract and reanalyze all positive results.
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TABLE 5. Description of PCR and extraction methods for detection of C. pneumoniae in vascular tissuea
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Jantos et al. (64) have also shown that the choice of DNA detection method is important. Coronary atherectomy specimens from 50 patients with CHD were examined for the presence of C. pneumoniae by two different PCR methods and by ISH. Specimens from four patients (8%) were positive by an omp1-based nested PCR method, specimens from two (4%) patients were positive by a single-step 16S rRNA-based PCR, and C. pneumoniae was not detected by ISH in any of the cardiovascular tissues tested. ISH was previously used by Ramirez et al. (113). The only culture-positive specimen in that study was also the only specimen that was positive by ISH.
Some authors have reported a higher prevalence of C. pneumoniae in more advanced atherosclerotic lesions when using immunological antigen detection techniques (30, 73). It is not yet known if this is due to a higher prevalence of C. pneumoniae in advanced atherosclerotic tissue or is due to a lower specificity because of the damaged blood vessels, since other methods have shown discordant results with a similar prevalence of C. pneumoniae in mild and advanced atherosclerosis. By means of PCR, Thomas et al. (138) correlated the prevalence of C. pneumoniae with the severity of atherosclerosis based on the Stary classification. The distribution of C. pneumoniae did not match the extent of severity of atherosclerosis since the prevalence was as high in mild lesions as in severe ones. Davidson et al. (25) found no difference in the severity of atherosclerosis in subjects with and without C. pneumoniae present in coronary arteries. Shor et al. (129) reported a strong association between atheromatous lesions and C. pneumoniae; however, minimal lesions were C. pneumoniae positive as often as severe lesions were.
Serologic analyses are not useful for predicting carriage of C. pneumoniae in the vascular wall (84), but several investigators have reported that it is rare with a vascular C. pneumoniae infection in C. pneumoniae-seronegative adult individuals (9, 82, 84, 112). Thus, at the best, serology may be used as a screening tool. PCR-based detection of C. pneumoniae DNA in peripheral blood mononuclear cells (PBMCs) may prove useful for identifying C. pneumoniae carriers and to measure the effect of antichlamydial therapy, since C. pneumoniae may be harbored in circulating white blood cells (8, 10, 12, 13, 15, 67, 86, 135, 148). However, the reported prevalence of C. pneumoniae in PBMCs has varied significantly, ranging from 9 to 59% of patients with CAD and from 17 to 46% of healthy blood donors (12, 13, 148). Therefore, systematic efforts to optimize and standardize the tests and test procedures are urgently needed. Standardized quantitative nucleic acid amplification methods for the detection of C. pneumoniae in circulating white blood cells and vascular tissue may enable better identification of individuals with persistent and vascular C. pneumoniae infection and improve the diagnostic tools in antibiotic treatment trials. It has not yet been shown that carriage of C. pneumoniae DNA in circulating white blood cells in humans represents a viable infection. In only one study of C. pneumoniae DNA in circulating white blood cells have the PCR-generated results been confirmed by another detection method (12).
The major assumption of many of the numerous seroepidemiologic studies is that the presence of anti-C. pneumoniae antibody implies the presence of the organism in the vascular system. The results of several studies have found that C. pneumoniae serology frequently does not correlate with identification of the organism by PCR or ICC in atheromas and C. pneumoniae DNA in circulating peripheral blood monocytic cells (7, 8, 17, 67, 84). Campbell et al. (17) did not observe an association between detection of C. pneumoniae in native or restenosed coronary arteries and MIF antibodies. Maass et al. (84) detected C. pneumoniae DNA by PCR in atheromas from 34 (26.6%) of 128 patients undergoing coronary surgery. There was no significant difference in the proportion of C. pneumoniae-positive and C. pneumoniae-negative patients who had detectable anti-C. pneumoniae IgG (100 and 81%, respectively) or IgA (38 and 46%, respectively) by MIF. An IgG titer of
16 was used to indicate seropositivity; the cutoff titer for IgA was not specified. The geometric mean titers also did not differ significantly. A subsequent study by the same group examined coronary arteries or occluded vein grafts from patients undergoing primary or repeat bypass surgery (7). Although C. pneumoniae was detected by culture and/or PCR in 19 and 18% of the native coronary arteries and grafts, respectively, there was no statistical difference between the IgG titers in the C. pneumoniae-positive or -negative patients.
Berger et al. (8) found C. pneumoniae DNA in 25% of atherosclerotic plaques and 20% of PBMCs in 130 patients who underwent surgery for carotid stenosis, aneurysm, or peripheral vascular disease. In addition to finding that the presence of C. pneumoniae in the plaque and PBMCs did not coincide, there was no correlation with IgG or IgA titers determined by MIF. Overall, 73% of the patients had an IgG titer of 64 and 68% had an IgA titer 64. There was no significant difference between the prevalence of antibody or titers of IgG or IgA in the patients who tested positive for C. pneumoniae DNA compared to those who were negative. Similar results were reported by Kaul et al. (67), who found C. pneumoniae DNA in PBMCs of 13 (43.3%) of 30 patients with CAD and 5 (26.3%) of 19 healthy age- and sex-matched blood donors. Only 8 (62%) of 13 PCR-positive patients with CAD and 3 (60%) of 5 PCR-positive blood donors were found to be seropositive by MIF (IgG 32).
The increasing interest in chronic C. pneumoniae infections has led to many new investigators and laboratories involved in C. pneumoniae research; the focus of most of these investigations has been on C. pneumoniae and atherosclerosis. Since the majority of the tests used are in-house developed, and the results from many of the studies are contradictory and confusing, standardization of the methods has been called for (14). Therefore, guidelines have recently been formulated to ensure that results as reproducible and comparable as possible are obtained (27).
Different detection methods have provided evidence that C. pneumoniae is present in a proportion of atherosclerotic blood vessels, but the true prevalence is still unknown because of divergent inter- and intralaboratory results, as a result of insufficient standardization of the methods used. In this paper, we suggest a gold standard that includes two independent C. pneumoniae-specific tests. Using this standard, we have calculated the prevalence of C. pneumoniae in atheromatous vascular tissue to be approximately 15% (Table 2). This percentage may be an underestimation of the true prevalence, but it may also be an overestimation as a result of poor validation of the methods used in most of the studies. With few exceptions (102, 147), the studies that have included controls indicate that the presence of C. pneumoniae (or some antigens of the bacterium) in atherosclerotic lesions is more frequent than in normal, nonatherosclerotic tissue, but the choice of control tissue has been criticized (134) and several major questions remain unresolved, for example, what is the true prevalence of C. pneumoniae in atheromatous tissue? What is the true prevalence of C. pneumoniae in normal, carefully matched, vascular tissue? What is the true prevalence of viable C. pneumoniae infection in such tissue? Is the higher prevalence detected by ICC compared with other techniques, including PCR (17, 43, 58, 66, 73), a result of higher sensitivity or lower specificity? Is the bacterium an innocent passenger, or is it involved in the initiation or progression of disease?
If C. pneumoniae is present in atherosclerotic vascular tissue, it should be emphasized that this suggests an association but does not establish an etiologic relationship between C. pneumoniae and atherosclerosis; it may simply represent a secondary event reflecting inflammation.
CLINICAL TREATMENT INTERVENTION STUDIES
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Although recent seroepidemiologic studies and evaluations of the performance of MIF and PCRs are creating considerable uncertainty regarding the existence of a causal relationship of infection with C. pneumoniae and CVD, several antibiotic trials have been published or are ongoing. The underlying rationale is that antibiotic treatments that are effective against C. pneumoniae may lower the risk for further cardiac events in patients with CVD by eradicating the organism from plaques, thus stabilizing the plaques (90). These studies fall into two categories, retrospective case-control studies of past antibiotic use and subsequent risk of MI and prospective, placebo-controlled treatment studies. However, even data on the microbiologic efficacy of treatment of respiratory infections due to C. pneumoniae are limited. None of the regimens being used in these trials have actually been evaluated or demonstrated to be effective in the eradication of C. pneumoniae from respiratory sites.
C. pneumoniae is susceptible in vitro to a wide range of antimicrobial agents, including macrolides, tetracyclines, and quinolones, that target either protein or DNA synthesis (Table 6) (39, 52, 53, 54, 119, 120, 121). Recently, Gieffers et al. (39) reported that the in vitro susceptibilities of five isolates of C. pneumoniae cultured from vascular tissue did not differ significantly from those of respiratory isolates. However, in vitro activity may not always predict in vivo efficacy. Anecdotal data suggest that prolonged therapy (i.e., at least 2 weeks) may be desirable since recrudescent symptoms have been described following 2-week courses of erythromycin and even after 30 days of tetracycline or doxycycline therapy (42, 51). Practically all pneumonia treatment studies evaluating new macrolides/azalides and quinolones presented or published to date have used serology alone for diagnosis, essentially limiting themselves to a clinical end point. In 1990, Lipsky et al. (81) described four patients with bronchitis and pneumonia, treated with a 10-day course of ofloxacin, who were retrospectively identified as having serologic evidence of C. pneumoniae infection (fourfold rise in IgG/IgM titer, single IgM titer of
16 or IgG titer of 512). All reportedly demonstrated marked clinical improvement. Based on the MICs of ofloxacin to three laboratory strains (1 to 2 µg/ml), the authors concluded that ofloxacin was effective in these patients since the MICs were lower than achievable serum levels. Subsequent treatment studies have followed this premise: if the patient has serologic evidence of infection and clinically improves, the organism has been presumed to be eradicated. Another example is the multicenter treatment study reported by File et al. (34) in 1997. They found a clinical cure rate of 98% among patients with serologic evidence of C. pneumoniae infection who were treated with levofloxacin compared to 93% of those treated with ceftriaxone and/or cefuroxime axitil, plus erythromycin or doxycycline, which was added to the treatment regimen at the investigator’s discretion. In the latter group, the response rate did not differ between patients who had erythromycin or doxycycline added to their treatment regimen and those who were treated with a cephalosporin alone. There was also no difference in the response rate among patients who had "definite" serologic evidence of infection, i.e., a fourfold rise in MIF IgG or IgM titer, compared to those who had "probable" infection, i.e., a single IgG titer of 512 or IgM titer of 32. The success of the cephalosporin regimens supports concerns about the specificity of the serologic criteria since these antibiotics have no or poor activity against Chlamydia spp. in vitro (50). A number of subsequently published treatment studies have claimed microbiologic eradication, despite the fact that cultures were not done (54). Conversely, the results of studies that have assessed microbiologic efficacy have frequently found that patients improve clinically despite persistence of the organism. |
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TABLE 6. Comparative in vitro activities of various antimicrobials against C. pneumoniaea
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Meier et al. (89) attempted to determine whether previous use of antibiotics decreased the risk of developing a first-time acute MI by examining a population-based database from the United Kingdom in a case-control study. Records from a total of 3,315 cases and 13,139 age- and sex-matched controls were examined. They found that cases were significantly less likely to have used tetracyclines or quinolones than controls. No effect was found for prior use macrolides (primarily erythromycin), penicillins, or cephalosporins. A history of antibiotic exposure meant any antibiotic use during an arbitrarily selected 3-year period prior to the date of the acute MI. Although the dose of antibiotic was included in the analysis, there was no consideration of the duration of treatment. The finding of a protective effect with tetracyclines, but not macrolides, was inconsistent since the in vitro activities of both classes of drug are similar (Table 6). There are no data suggesting that tetracyclines are more effective than macrolides in treatment of C. pneumoniae infection. Only eight cases received quinolones, which included ofloxacin, ciprofloxacin, and norfloxacin. The last two drugs are relatively inactive against C. pneumoniae in vitro (Table 6), and ciprofloxacin has inadequate microbiologic efficacy in the treatment of genital C. trachomatis infection (114). A subsequent study from the University of Washington, using a database from a large health maintenance organization, found that use of erythromycin, tetracycline, or doxycycline during the previous 5 years was not associated with a lower risk of a first MI (60). The population included 1,796 patients and 4,883 controls. Unlike the study of Meier et al. (89), an attempt was also made to assess risk in relation to the duration of treatment. Patients who were treated for
29 days with any of the drugs had the same risk as those who were treated for a shorter duration or had received no antibiotics. There are now three small, randomized controlled secondary prevention studies published in the literature (44, 46, 47, 96), and another five studies which are larger, are ongoing (Table 7) (24, 28, 45, 63). The rationale for these studies is that treatment of a putative, active C. pneumoniae infection will reduce the risk of further cardiac morbidity and mortality in patients who have had an acute MI. Azithromycin at various doses and durations of therapy has been the drug of choice in six of these trials. Azithromycin has in vitro MICs similar to those of erythromycin, but it has a half-life in tissue of
5 days, which allows for extended dosing regimens (28). The other macrolide used is roxithromycin, which has in vitro activity similar to erythromycin (121). |
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TABLE 7. Summary of placebo-controlled clinical trials for secondary prevention of CVD
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8). Patients who had IgG titers of 64, which was considered to indicated "persistent" infection, were randomized to receive azithromycin at 500 mg/day for 3 or 6 days. They found that patients receiving azithromycin demonstrated a decrease in their MIF IgG titers and had a lower risk for adverse cardiovascular events than did seropositive patients who were not treated. Subsequent studies have not found a significant effect on cardiovascular mortality or morbidity or serologic markers of C. pneumoniae infection. A larger, subsequent study reported that treatment with azithromycin, 500 mg/day for 3 days followed by 500 mg/week for 3 months, was not associated with reductions in ischemic events, including cardiovascular death, nonfatal MI, stroke, and unstable angina (96). Patients were enrolled if they were seropositive with an IgG of 16 in MIF. At the end of the 24-month follow-up period, they observed reductions in inflammatory markers, including CRP, interleukin-6, and tumor necrosis factor alpha, but no effect on anti-C. pneumoniae antibody titers. Gurfinkel et al. (47) also failed to find a significant difference in clinical end points (cardiac death, MI, or severe recurrent ischemia) in patients with unstable angina or non-Q-wave MI at 90 or 180 days after treatment with roxithromycin 150 mg twice a day for up to 30 days or placebo. Unlike the previous studies, C. pneumoniae seropositivity was not an enrollment criterion. Anti-C. pneumoniae IgG titers did not change with therapy and did not differ between the treatment and placebo groups. Treatment with roxithromycin also appeared to decrease the levels of CRP compared to placebo.
The effect of antibiotic treatment on anti-C. pneumoniae antibody and inflammatory markers has been reported in several small studies that did not specifically look at CVD end points. Jackson et al. (61) randomized 88 patients following percutaneous coronary revascularization to receive either azithromycin, 500 mg/day for 2 days then 250 mg/day on days 3 through 28, or placebo. They found that although azithromycin was well tolerated, there was no effect on either IgG or IgA antibody titers. Similar findings were also reported by Sinisalo et al. (130), who found that treatment with doxycycline, 100 mg twice a day for 4 months, had no effect on anti-C. pneumoniae MIF antibody titers in men with angiographically confirmed CHD and prior bypass surgery. There were no significant differences in levels of CRP, cholesterol, high-density lipoprotein, and low-density lipoprotein between patients who received doxycycline or placebo. Torgano et al. (139) randomly assigned 97 patients with CVD to receive either clarithromycin, 500 mg twice a day for 30 days (if they were seropositive for H. pylori regardless of C. pneumoniae antibody status) or 500 mg twice a day for 14 days (if they were seropositive only for C. pneumoniae [MIF IgG titer, 64]), or placebo. They found a significant reduction in the geometric mean IgG titer in the treated patients and significantly reduced serum fibrinogen levels in patients who were treated with 30-day, but not 14-day, courses of clarithromycin. Semaan et al (127) treated 40 patients who had documented CVD and an anti-C. pneumoniae MIF IgG titer of >16 and 20 controls who had normal coronary arteries with azithromycin, 500 mg/day for 3 days then twice weekly for 3 months, or placebo. They found that azithromycin treatment had no effect on the levels of soluble markers of endothelial activation, intercellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin, in plasma.
As of this writing, there are five ongoing, larger, randomized controlled secondary prevention studies, which are summarized in Table 7, although some details are not available for all the studies. Four of these studies are using weekly azithromycin regimens for periods of 2 weeks to 12 months. The remaining study is using daily gatifloxacin for 18 months (45). Gatifloxacin is a quinolone with MIC and MBC for 90% of isolates (MIC90 and MBC90) of 0.25 mg/ml against recent clinical isolates of C. pneumoniae (54), but there are no data on the microbiologic efficacy of this drug. Three studies are screening patients for anti-C. pneumoniae IgG by MIF, using different titers as enrollment criteria. In addition, each study uses different cardiac entry criteria, including previous MI or bypass surgery, acute MI at least 6 weeks before enrollment, and severe CHD waiting for bypass surgery. The Weekly Intervention with Zithromax for Atheroscelrosis and Related Diseases (WIZARD) study is using an IgG titer of 16 (28). Patients will then be randomized to receive azithromycin, 600 mg four times/day for the first 3 days then 600 mg once weekly, or placebo. Patients will be enrolled regardless of their serologic status in the Azithromycin Coronary Events Study (ACES) and Might Azithromycin Reduce Bypass-List Events? (MARBLE) studies (45, 63). In the ACES study, patients will be treated with azithromycin, 600 mg weekly for 1 year, or placebo. Patients in the MARBLE study will be treated with azithromycin 600, mg/day for 3 days followed by 600 mg/week for 3 months. The South Thames Antibiotics in Myocardial Infarction and Angina study (STAMINA) proposes to treat patients who have serologic evidence of C. pneumoniae and H. pylori with azithromycin, an amoxicillin-based regimen, or placebo (45). The end points include the effect on inflammatory markers and cardiovascular events. None of these studies have reported any data as yet. The WIZARD study extended the enrollment period for another year to recruit more patients.
Reasons for failure to find an effect of antibiotic treatment on adverse cardiac outcome in two of the initial three intervention studies include low power due to small numbers, inadequate dosing and duration of treatment, or poor positive predictive value of C. pneumoniae serology. In terms of dosage and duration of treatment, none of the antibiotic regimens used previously or being used currently has been evaluated or demonstrated to be effective in eradication of C. pneumoniae from the respiratory tract or any other site. Ironically, the only study that found a significant reduction in adverse cardiac outcome used an azithromycin regimen, 500 mg/day for 3 or 6 days, that was the least likely to be effective (44). As described previously, 1.5 g of azithromycin over 5 days had only 70% efficacy in eradicating C. pneumoniae from the nasopharynx of adults with pneumonia (120). Furthermore, the weekly dosing regimens of azithromycin used in the ACADEMIC study and the ongoing intervention studies described previously may result in long periods of subinhibitory levels of drug, which may lead to the development of resistance. This has been demonstrated to occur with Streptococcus pneumoniae and S. pyogenes after azithromycin treatment (36) and could potentially occur with C. pneumoniae. Extrapolating from the persistently infected cell model (77, 79), one would also expect that these weekly azithromycin regimens would not be effective in eradicating chronic C. pneumoniae infection, if present. The reductions in inflammatory markers reported in several studies (47, 96) may be secondary to anti-inflammatory activity of macrolides and tetracyclines (126). Roxithromycin has been demonstrated to have very potent anti-inflammatory activity in vitro (126). Finally, as discussed for the seroepidemiologic studies, there are no reliable serologic markers for chronic or persistent C. pneumoniae infection. In reality, this means that we really cannot determine who is infected and who is not. It also means that we cannot assume that any effect seen is due to successful treatment or eradication of C. pneumoniae.
The MARBLE study proposes a slightly different approach. As stated above, patients with severe CAD waiting for bypass surgery will be treated with azithromycin or placebo for 3 months (45). These patients can typically wait for up to 12 months for surgery. At the time of operation, samples of coronary artery will be examined for the presence of C. pneumoniae by PCR. Melissano et al. (91) reported a similar pilot study where 32 patients were treated with either roxithromycin, 150 mg twice a day, or placebo for 17 to 35 days before undergoing carotid endarterectomy. Using a seminested PCR, C. pneumoniae DNA was detected in plaques of 5 (31.3%) of 16 treated patients compared to 12 (75%) of 16 who received placebo (P = 0.034). The authors claimed that these results demonstrated that treatment with roxithromycin was effective in eradicating C. pneumoniae from the plaques. However, since the pretreatment status was unknown and given the problem observed with performance of PCR, one cannot really assume that the lower prevalence of C. pneumoniae DNA in the treatment group was due to successful eradication.
Recently Gieffers et al. (40) treated two patients who had acute unstable angina with azithromycin, 500 mg/day for 3 days followed by 500 mg on days 14 and 16 after coronary angiography. PBMCs obtained before treatment were positive for C. pneumoniae DNA, but the organism was isolated by culture from PBMCs obtained on day 28 after angiography. These results are not totally unexpected since the dose and duration of therapy with azithromycin were probably suboptimal.
Rabbit and mouse models of atherogenesis have been used to determine if infection with C. pneumoniae can induce plaque formation. Fong et al. (35) and Muhlestein et al. (95) have demonstrated that C. pneumoniae can cause atheromatous lesions in the aorta and increase intimal thickening in 30 to 40% of New Zealand White rabbits after intranasal inoculation. In mice, C. pneumoniae disseminates after intranasal inoculation and can be isolated from the lungs, spleen, and buffy coats (145). Infection with C. pneumoniae has also been demonstrated to cause increasing fatty-streak lesion formation in apolipoprotein E (ApoE)-deficient mice, which develop these lesions spontaneously (123). Other mouse models, including BALB/c mice, which develop atherosclerotic lesions when fed an atherogenic diet, have not demonstrated a consistent association of C. pneumoniae infection with enhancement of atherosclerosis (145). Treatment of acute C. pneumoniae infection in rabbits with high-dose azithromycin for 10 weeks was associated with reduction of intimal thickening but did not appear to eradicate evidence of the organism as demonstrated by ICC (35, 95). Rothstein et al. (123) reported that treatment of ApoE-deficient mice 4 to 5 weeks after C. pneumoniae infection with azithromycin had no effect on the size of the atherogenic aortic lesions. In addition, azithromycin treatment did not affect the size of lesions in sham-infected mice or the persistence of C. pneumoniae in the aorta, lung, or spleen, and it did not affect antibody titers. These results seem to support the experience with rabbits, which found an effect with treatment of acute but not established infection. One implication of these data for human secondary prevention trials is that antibiotic treatment may not be beneficial, since C. pneumoniae infection in patients with CVD, who are being enrolled in these studies, is most probably not acute.
CONCLUSIONS
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The role of infection and specifically, C. pneumoniae remains controversial and unresolved. It now appears that some of the inconsistency of the results from study to study may be due, in part, to lack of standardized methods. Although initial seroepidemiologic studies demonstrated a significantly increased risk of adverse cardiac outcome in patients who were seropositive, subsequent prospective studies found either small or no increased risk. In addition to the lack of consistent serologic criteria, recent evaluations have demonstrated inherent problems with performance of the most widely used serologic methods. Most importantly, we do not have a reliable serologic marker for chronic or persistent C. pneumoniae infection. Similarly, the results of studies demonstrating the presence of C. pneumoniae have also been extremely variable. As with the seroepidemiologic studies, the PCR and ICC methods used are not standardized. The results of recent studies suggest that laboratories using the same methods with the same specimens may obtain different results. However, it is clear that C. pneumoniae is present in atherosclerotic plaque, since it has been isolated by culture in a limited number of studies, albeit at a relatively low frequency. It has been suggested that the results of the larger treatment studies will help to define or confirm the role of C. pneumoniae in CVD. Even if a reduction in adverse events is seen, one cannot assume that it was due to treatment of C. pneumoniae.
FOOTNOTES
* Corresponding author. Mailing address: Department of Pediatrics, Box 49, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203-2098. Phone: (718) 245-4075. Fax: (718) 245-2118. E-mail: mhammerschlag{at}pol.net.
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0893-8512/01/$04.00+0 DOI: 10.1128/CMR.15.1.1-20.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
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