1. health and fitness
2. disease

CHLAMYDIA PNEUMONIAE AND ATHEROSCLEROSIS
Facts or fiction
Hans F. Berg
ISBN: 90-77595-68-6
© Hans F. Berg, Breda, 2004
Printed by: Optima Grafische Communicatie, Rotterdam
Cover design: Toren van Babel II (aquarel op papier, 2003) by Herman Kuypers
“Feiten en fictie treffen elkaar in mijn werk. Het vertrouwde van het alledaagse, in mijn
geval vooral stoelen en tafels, door middel van bijvoorbeeld standpunt, ordening,
recombineren of ontleden, verbijzonderen tot archetypische beelden. Mijn kennis van en
de fascinatie voor perspectief zijn daarbij onmisbaar”. Herman Kuypers, Tilburg
Publication of this thesis was financially supported by Abbott BV.
VRIJE UNIVERSITEIT
CHLAMYDIA PNEUMONIAE AND ATHEROSCLEROSIS: FACTS OR FICTION
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. T. Sminia,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de faculteit der Geneeskunde
op vrijdag 1 oktober 2004 om 13.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Hans Floris Berg
geboren te Breda
promotor:
prof.dr. C.M.J.E. Vandenbroucke-Grauls
copromotor:
dr. J.A.J.W. Kluytmans
Aan Noortje
Aan mijn ouders
CONTENTS
Contents
Chapter 1
Introduction
9
Chapter 2
Correlation between detection methods of Chlamydia pneumoniae
in atherosclerotic and non-atherosclerotic tissues
23
Diagn Microbiol Infect Dis 2001;39:139-143
Chapter 3
Extraction of Chlamydia pneumoniae-DNA from vascular tissue
for use in PCR: an evaluation of four procedures
35
Clinical Microbiology and Infection 2003;9:135-139
Chapter 4
Effect of clarithromycin treatment on Chlamydia pneumoniae in
vascular tissue of patients with coronary artery disease: a
randomised, double-blind, placebo-controlled trial
45
Submitted
Chapter 5
Effect of clarithromycin on inflammatory markers in patients with
atherosclerosis
61
Clinical and Diagnostic Laboratory Immunology 2003;10(4):525528
Chapter 6
Treatment with clarithromycin prior to coronary artery bypass graft
surgery does not prevent subsequent cardiac events
73
Submitted
Chapter 7
Emergence and persistence of macrolide resistance in
oropharyngeal flora after clarithromycin therapy: a randomised,
double-blind, placebo-controlled study
87
Submitted
Chapter 8
Summary and conclusions
103
Samenvatting en conclusies
117
Publicaties
127
Dankwoord
131
Curriculum Vitae
134
CHAPTER
GENERAL INTRODUCTION AND OUTLINE OF THE THESIS
1
CHAPTER 1
INTRODUCTION
This thesis describes investigations that were performed to gain more insight into the
role of Chlamydia (C.) pneumoniae in relation to atherosclerosis. In this introduction the
current knowledge of the pathogenesis of atherosclerosis, the role of C. pneumoniae,
and the effects of antibiotic treatment are described.
Atherosclerosis
Atherosclerosis is a major cause of morbidity and mortality in both industrialised and
developing countries, with a broad spectrum of clinical manifestations. In the year 2000
for example, coronary heart disease caused more than 1 of every 5 deaths in the United
States and was therefore the leading cause of death among men and women. Acute
myocardial infarction was diagnosed in 540.000 Americans in the same year1.
Atherosclerosis is also a major cause of physical disability, particularly in the rapidly
growing population of elderly persons2,3. The prevention of coronary events and the
maintenance of physical functioning are major challenges in preventive care.
The process of atherogenesis begins at an early age4. The earliest type of lesion,
the so-called fatty streak, is common in infants and young children5. The fatty streak is a
pure inflammatory lesion6, consisting of monocyte-derived macrophages and specific
subtypes of T-lymphocytes. The initiation of atherosclerosis is characterized by
remodeling of the arterial intima. The arterial wall thickens, which is compensated by
gradual dilation, while monocytes and macrophages accumulate. This is accompanied
by the release of hydrolytic enzymes, cytokines, chemokines, and growth-factors. As the
atherogenesis process advances, foam-cells are formed by accumulation of cholesterol
in macrophages. Finally an advanced and complicated lesion evolves: the fibrous
plaque. In this phase the artery can no longer compensate by dilatation. The endothelial
surface of the lesion is damaged; the plaque becomes vascularized leading to
hemorrhage and thrombosis formation. Finally the artery can become completely
obstructed.
Research on the pathogenesis of atherosclerosis has focused for decades on
several now well known risk factors, which include smoking, hypercholesterolaemia,
diabetes mellitus, hypertension, and a positive family history of cardiovascular disease.
However, these risk factors do not fully account for all cardiovascular patients. Up to
30% of patients presenting with myocardial infarction for example have none of these
10
GENERAL INTRODUCTION
risk factors7. Furthermore, in some populations, the marked frequency of classical risk
factors is not associated with a high prevalence of cardiovascular disease8. Therefore
the search for novel potential risk factors continues.
The hypothesis that atherosclerosis may be a 'response-to-injury' was postulated
more than a century ago by Von Rokitansky9 and Virchow10. The involvement of
inflammatory response including inflammatory cells (macrophages and T lymphocytes)
and cytokines in all stages of the atherosclerotic process suggests a central role for
inflammation in atherosclerosis11. The idea of infections playing a role was proposed
over a century ago by investigators in Europe, but was championed in North America by
Osler12 and Billings13 in the early part of last century. However, evidence that infection
might induce atheromatous change was not found until the 1970’s, when Fabricant et al.
infected germ-free chickens with an avian herpesvirus, producing arterial lesions that
resembled atherosclerosis in humans14. Since then, a number of microbes have been
implicated in the etiology of atherosclerosis, including C. pneumoniae.
Chlamydia pneumoniae
Chlamydiae were originally classified in the order Chlamydiales, with only one family, the
Chlamydiaceae, and one genus, Chlamydia. This genus contained four species: C.
pecorum, C. psittaci, C. trachomatis, and C. pneumoniae. Recently a new classification
is proposed based on DNA sequence analysis by Everett et al.15 (figure 1). In this
reclassification the family Chlamydiaceae, is divided into two genera, Chlamydia and
Chlamydophila. Two new species, Chlamydia muridarum and Chlamydia suis, are added
to Chlamydia trachomatis in the genus Chlamydia. Chlamydophila contains the current
species Chlamydophila pecorum, Chlamydophila pneumoniae and Chlamydophila
psittaci. Three new Chlamydophila species are derived from Chlamydia psittaci:
Chlamydophila abortus, Chlamydophila caviae, and Chlamydophila felis. Furthermore,
three
new
non
Parachlamydiaceae,
Chlamydiaceae
and
families
Waddliaceae.
have
An
been
impressive
added,
Simikaniaceae,
number
of
eminent
chlamydiologists, are opposed to the new classification and their objections were
published in a letter16. The main criticisms were that there is insufficient reason to divide
the former Chlamydiaceae into 2 new genera and that it had taken a long time to get
clinicians used to Chlamydia and would be confusing for them. In this thesis the old
classification name Chlamydia pneumoniae was used, to prevent confusion.
11
CHAPTER 1
Figure 1. The new classification of the order Chlamydiales.
Order
Family
Genus
Species
Chlamydiales
Chlamydiaceae
Chlamydia
trachomatis
suis
muridarum
Chlamydophila
pneumoniae
psittaci
abortus
felis
caviae
pecorum
Simkaniaceae
Simkania
negevensis
Parachlamydiaceae
Parachlamydia
acanthamoeba
Neochlamydia
hartmanellae
Waddliaceae
Waddlia
chondrophila
C. pneumoniae has been recognized as a human pathogen since 1986 and is
now known to be a common cause of respiratory tract infection, causing bronchitis,
sinusitis, pharyngitis and accounts for over 5% of all cases of community-acquired
pneumonia17. Infection is usually associated with relatively mild disease. In addition to
the acute manifestations of infection, C. pneumoniae has been implicated as an
etiological
agent
in
the
development
of
several
chronic
diseases.
Besides
atherosclerosis, also adult-onset asthma and multiple sclerosis are mentioned18,19.
C. pneumoniae, C. trachomatis, and C. psittaci are known to cause disease in
humans. Unlike C. trachomatis, C. pneumoniae is not transmitted sexually and unlike C.
psittaci, it has no animal reservoir20. Humans are the only known reservoir of C.
pneumoniae.
Like all Chlamydiaceae, C. pneumoniae is an obligate intracellular organism. The
presence of a lipopolysaccharide cell wall, DNA and RNA, and the ability of the
12
GENERAL INTRODUCTION
organisms to replicate by binary fusion classify them as Gram-negative bacteria21. Their
developmental life cycle occurs in the cytoplasm of a host cell. The life cycle of all
chlamydiae has two distinct phases: a smaller extracellular non-replicating infectious
phase called elementary body (EB) and a larger intracellular non-infectious replicating
phase, called reticular body (RB). After attachment to host cells, elementary bodies enter
the cell, probably by endocytosis, and differentiate into reticulate bodies. Reticulate
bodies replicate, using the host cell energy, and form inclusions. Prior to cell lysis and
release from the host cell reticular bodies retransform into elementary bodies22. This
biphasic life cycle lasts for 48 to 72 hours.
Different C. pneumoniae isolates have 94 to 100% DNA homology with each
other but less then 10% with C. trachomatis or C. psittaci23. Furthermore, elementary
bodies of C. pneumoniae are pear-shaped, with a large periplasmic space, clearly visible
on electron microscopy (figure 2), whereas the elementary bodies of C. trachomatis, C.
psittaci and C. pecorum are apple-shaped.
Figure 2. Elementary body of Chlamydia pneumoniae visible on electron microscopy (x80.000).
Most of our current understanding of the epidemiology of C. pneumoniae
infection derives from serologic studies using the microimmunofluorescence assay
(MIF). These studies indicate that C. pneumoniae infection is common throughout the
world. Seropositivity is low in early childhood (< 5 years), rising rapidly during school
years. At the age of 20 years, approximately 50% of individuals will have detectable
antibodies. Seroprevalence continues to rise, albeit at a slower pace, in adulthood and
reaches 75% in the elderly population20,22. This high antibody prevalence in later life
suggests that reinfection with the organism is common. Seroprevalence is approximately
13
CHAPTER 1
equal in both sexes at < 15 years of age, but seropositivity rates in adults are higher in
men than in women.
While C. pneumoniae infection is an endemic disease, local epidemics are
described in nursing homes24, schools25, and military institutions26,27. There is no clear
seasonal variation in disease, but retrospective serological studies suggest that
community-wide epidemics occur in 4-6 year cycles28,29.
Clinical manifestations of C. pneumoniae infection range from asymptomatic to
mild disease, and very rarely severe life threatening infection occurs. Pneumonia and
bronchitis are the most frequently observed manifestations of C. pneumoniae infection.
Multiple studies have shown that C. pneumoniae is responsible for 5-20% of cases of
community-acquired pneumonia (CAP).
Diagnosis of C. pneumoniae infections remains difficult. Several techniques are
currently employed:
Serologic testing. The MIF test is considered to be the standard serologic assay for
determination of Chlamydia seropositivity30. The test is able to differentiate among the
IgG, IgA, and IgM antibody classes. However, it has some major drawbacks: it is timeconsuming and requires a very experienced microscopist for interpretation of the slides.
Furthermore, the specificity of MIF has been questioned, as cross-reactions among the
major outer membrane proteins of different Chlamydia species were reported31,32.
Because of these problems related to MIF, enzyme-linked immunosorbent assays
(ELISAs) and enzyme immunoassays (EIAs) were commercially developed; they are
relatively simple to perform, are less time-consuming, are more objective because of the
photometrical reading of the results, and are easier to standardize.
Isolation. C. pneumoniae can be isolated in cell culture. The organism is fastidious and
slow growing. Therefore isolation is not usually attempted for documentation of infection
in the clinical setting but can be useful for research purposes. However, primary isolation
from clinical specimens like atheromatous lesions is very difficult.
In addition, direct detection of organisms is possible by electron microscopy (EM),
immunohistochemistry (IHC) staining, and polymerase chain reaction (PCR).
IHC staining. Antigen detection methods with use of genus- or species-specific
antibodies labeled with peroxidase or a fluorescent marker are commonly used to
identify Chlamydia in tissue. Like EM, these techniques may allow localization of the
organisms in specific cells and areas. Considerable difficulty has been experienced with
IHC because of non-specific background staining.
14
GENERAL INTRODUCTION
PCR testing. PCR techniques using various C. pneumoniae-specific primers have been
developed and used for detection of the organism in pharyngeal swabs, bronchoalveolar
lavage, and sputum specimens. Compared to cell culture PCR is much more sensitive
and highly specific. It has the potential to detect a single organism. This method has also
been used to detect C. pneumoniae in pathologic tissue specimens. However, to date,
there are no standardized PCR or other nucleic acid amplification methods for the
detection of C. pneumoniae.
Erythromycin, tetracycline, and doxycycline are active in vitro against C.
pneumoniae and are recommended as first-line therapeutic agents. Newer macrolides,
like clarithromycin, are also active in-vitro and achieve high intracellular concentrations.
These agents are better tolerated than erythromycin and therefore may be considered
as either first-line agents or as alternatives to erythromycin and the tetracyclines for
treatment of infections believed to be due to C. pneumoniae. The organism is not
susceptible to penicillin, ampicillin, or sulfa drugs33.
The link between atherosclerosis and C. pneumoniae
The initial study indicating a potential link between atherosclerosis and C. pneumoniae
infection was performed in Finland and showed that patients with coronary artery
disease (CAD) were more likely to have detectable antibodies to C. pneumoniae34. This
study examined sera from 40 men with acute myocardial infarction (AMI), 30 men with
CAD and 41 matched controls. Serologic evidence of previous infection (C. pneumoniaespecific IgG>128 and/or IgA>32) was found in 68% of patients with AMI (OR 11.92,
95%CI 3.65 - 40.91), 50% with chronic CAD (OR 4.86, 95%CI 1.46 - 16.67) and in only
17% of population-matched controls. Since this initial report, several investigators from
various countries have confirmed these seroepidemiological findings.
In 1993 Shor, a South African pathologist, noticed unusual pear-shaped
structures in coronary artery atheroma on scanning electron microscopy of autopsy
specimens. Using IHC and PCR techniques, Shor and the study group of Grayston were
able to confirm that these structures were the elementary bodies (EB) of C.
pneumoniae35,36. Since then, morphologic and microbiologic evidence for the presence
of C. pneumoniae has been found in a wide variety of diseased arteries. The organism
was identified in vascular tissue from patients with cardiovascular disease by electron
microscopy, PCR, and IHC. Sporadically, C. pneumoniae has also been isolated by
culture from atheromatous vascular specimen by some investigators: reports of isolation
15
CHAPTER 1
from a carotid endarterectomy specimen, the coronary artery of a recipient’s heart
removed prior to heart transplantation, and, in one report, coronary artery specimens
from 16% (11/70) of patients undergoing coronary artery bypass graft surgery37-39.
More direct evidence of causality as well as insight into potential pathogenic
mechanism was provided by animal experimental models. Results of studies using
ApoE-deficient knockout mice, which spontaneously develop atherosclerosis on a
normal diet, indicated that C. pneumoniae can be detected in aortic plaque specimen
after intranasal inoculation. C. pneumoniae seems to disseminate systematically after
respiratory infection, and the organism persists in aortic plaque for at least 20 weeks
following infection. The infection appears to accelerate the progression of the
atherosclerotic process in these animals. Studies in New Zealand white rabbits, which do
not typically develop atherosclerosis on a normal diet, found that animals infected
intranasally with C. pneumoniae later showed changes in the aorta consisting of intimal
thickening or fibrolipid plaques, and that C. pneumoniae could be detected in those
plaque specimens by IHC studies40-43.
In another study in New Zealand white rabbits, which were fed a slightly
cholesterol-enhanced diet to enhance atherosclerotic lesion development, were
inoculated intranasally with either C. pneumoniae or saline. The animals were
randomized to a 7-week course of treatment with azithromycin or to no treatment.
Infected, untreated animals had significantly larger areas of plaque involvement of the
thoracic aorta than those seen in uninfected controls. In contrast, the infected and
treated rabbits did not demonstrate this increase. This suggests that treatment with
azithromycin might prevent the infection-induced acceleration in atherosclerosic plaque
development44.
On the basis of these results, it was anticipated that infection with C. pneumoniae
might prove a novel treatable risk factor for atherosclerosis, with relevance comparable
to that of Helicobacter pylori in peptic ulcer disease45.
In 1997, Gupta and colleagues46 published the results of a small clinical trial in 60
survivors of acute myocardial infarction that had persistently elevated anti-C.
pneumoniae IgG titers ((MIF) >1:64). These individuals had been randomized to receive
a 3-day course of azithromycin (500mg/d), two such courses 3 months apart, or placebo.
Patients who received azithromycin showed a reduction in acute coronary events from
28% to 8% compared to those who received placebo (p=0.04, RR 0.27, 95% CI 0.06 0.95), but there was no additional benefit in receiving an additional course of therapy.
16
GENERAL INTRODUCTION
The results of this trial generated considerable interest and encouraged further studies
on the relation of C. pneumoniae and atherosclerosis.
Outline of this thesis
To gain more insight in the association between C. pneumoniae and atherosclerosis, we
initiated several projects.
Laboratory techniques play a crucial role in studies of the association of C.
pneumoniae and atherosclerosis. Therefore, the diagnostic assays were first validated.
Because discrepancies between results of different detection methods are often
reported, the correlation between two frequently used detection methods for C.
pneumoniae, i.e. PCR and IHC, is assessed in chapter 2. To optimise the detection of C.
pneumoniae by PCR, four procedures for C. pneumoniae-DNA extraction from vascular
tissue were evaluated, with respect to the yield of recovered DNA. The results are
presented in chapter 3.
Up to now no studies have been reported in which the effect of treatment on the
presence of C. pneumoniae in vascular tissue was investigated. In a placebo-controlled,
double blind, randomised intervention trial, we investigated whether C. pneumoniae
could be eradicated from vascular tissue by a short course of clarithromycin. The effect
of claritrhomycin was evaluated by IHC, real time PCR and LCx PCR (chapter 4).
Stabilisation of atherosclerotic plaques might be achieved by any anti-inflammatory
treatment regimen. There is much speculation about the anti-inflammatory potential of
macrolides, in particular of clarithromycin, independently of their established role as
antimicrobial agents. Therefore, the effect of clarithromycin on inflammatory markers is
studied in chapter 5. Because it has been hypothesized that antibiotic treatment effective
against C. pneumoniae might lower the risk for further cardiac events in patients with
severe atherosclerosis, the effect of treatment with clarithromycin prior to cardiac surgery
on clinical outcome and prognosis was determined in a two-year follow-up of these
patients (chapter 6).
The use of macrolide antibiotics like clarithromycin raises serious concern about
the development of macrolide resistance. Resistance to macrolide antibiotics, like
clarithromycin, is indeed increasing. Therefore, the effect of clarithromycin on the
development of resistance in the oropharyngeal and nasopharyngeal flora in the patients
described in chapter 7 was quantified (Chapter 7).
Finally, in chapter 8 the summary and conclusions are presented.
17
CHAPTER 1
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18
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20
GENERAL INTRODUCTION
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21
CHAPTER
2
CORRELATION BETWEEN DETECTION METHODS OF CHLAMYDIA
PNEUMONIAE IN ATHEROSCLEROTIC AND NON-ATHEROSCLEROTIC
TISSUES
B Maraha, HF Berg, GJ Scheffer, A van der Zee, A Bergmans, J Misere, JAJW Kluytmans, MF
Peeters
Diagn Microbiol Infect Dis 2001;39:139-143
CHAPTER 2
SUMMARY
Polymerase chain reaction (PCR) and immunohistochemistry (IHC) have been used to
detect Chlamydia (C.) pneumoniae in vascular tissues. Discrepancies between the
results of these two methods have frequently been reported. However, the correlation
between PCR and IHC has not been analyzed yet. This study assesses the correlation
between the detection of C. pneumoniae by PCR and IHC in 45 atherosclerotic and 50
non-atherosclerotic tissues specimens. Also, the presence of Mycoplasma (M.)
pneumoniae in these 95 specimens was investigated. Correlation was found between
the detection of C. pneumoniae by PCR and IHC in the atherosclerotic tissues. Both
tests were positive in 10 specimens and negative in 17 specimens (p=0.003). There was
no significant correlation between PCR and IHC in non-atherosclerotic specimens
(p=ns). M. pneumoniae was detected, by PCR, in one atherosclerotic specimen. The
results show correlation between PCR and IHC in the detection of C. pneumoniae in
atherosclerotic tissues and support the specificity of the association between C.
pneumoniae and atherosclerosis.
24
CORRELATION BETWEEN DETECTION METHODS OF C.PNEUMONIAE
INTRODUCTION
Using polymerase chain reaction (PCR) and immunohistochemistry (IHC), the
presence of Chlamydia (C.) pneumoniae has been demonstrated in atherosclerotic
tissues, but in control vascular tissues this pathogen has been found less frequently1.
These findings indicate that C. pneumoniae may play a role in the pathogenesis of
atherosclerosis.
In addition to C. pneumoniae, several microorganisms have been postulated as
possible risk factors for atherosclerosis2,3. The association between atherosclerosis and
both cytomegalovirus and Helicobacter pylori has been extensively studied2. However,
till now only one report on the presence of Mycoplasma (M.) pneumoniae in vascular
tissues has been generated4.
The microimmunofluorescence test (MIF), PCR and IHC have been used to
explore the association between C. pneumoniae and atherosclerosis. Discrepancies
among the results obtained by these methods have frequently been found5-7. Nothing,
however, is known about the correlation between the results of these detection methods.
The present study assesses the correlation between the detection of C.
pneumoniae by PCR and IHC. Also, the correlation between C. pneumoniae serology
and the detection of this pathogen, by PCR and IHC, in vascular tissue specimens was
analyzed. M. pneumoniae-PCR was performed to detect M. pneumoniae in atherosclerotic and non-atherosclerotic tissues.
MATERIALS AND METHODS
Specimens were collected from 95 patients undergoing cardiac surgery. Fortyfive atherosclerotic specimens and 50 non-atherosclerotic tissue specimens (aorticpunchs) were collected. Each specimen was divided into two portions; one for PCR and
one for IHC. The PCR portion was transported to the laboratory in a Tris-EDTA buffer
containing 0.5% sodium dodecyl sulfate. In the laboratory, specimens were stored at
minus 200C. The IHC portion was fixed in 10% phosphate-buffered formalin. In addition,
sera were obtained from all patients to measure the level of IgG antibodies against C.
pneumoniae. Specimens were evaluated blind. Patients were not examined to assess
the presence of C. pneumoniae clinical disease.
25
CHAPTER 2
Polymerase chain reaction
DNA extraction from specimens was performed using the QiAmp DNA minikit
(Qiagen, CA, USA). For the PCR template, 5 µL of 200 µL elution sample was used.
Detection of C. pneumoniae DNA was carried out by 16S rRNA gene-based
PCR, using the primers CpnA: 5’-TGA CAA CTG TAG AAA TAC AGC-3’ and CpnB: 5’CGC CTC TCT CCT ATA AAT-3’ as described previously8.
Detection of M. pneumoniae was performed by PCR assay based on the P1
adhesin gene using the primers Pn1: 5’-GCC ACC CTC GGG GGC AGT CAG-3’ and
Pn2: 5’-GAG TCG GGA TTC CCC GCG GAG G-3’ as described previously4,9.
To minimize the risk of contamination, strict PCR-anti-contamination precautions,
such as the use of UDG/dUTP, were taken to prevent carry-over of previous PCR
reactions. Sample processing and the PCR assay were performed in separate rooms
and a negative control was processed with every 5 samples. To control for inhibition of
the PCR reaction, a second sample of each specimen was spiked with target DNA (C.
pneumoniae or M. pneumoniae). Inhibited PCR samples were retested using 2.5 µL of
specimen. PCR products were visualized after electrophoresis in 2% ethidium bromidestained agarose gels.
To confirm positive results, 3 µL of the PCR product was spotted and hybridized,
with the 21 bp 5’-biotinylated probe Cpneu-B:5’ GAC ACA CGT ACA ATG GTT-3’ in the
case of C. pneumoniae-PCR and with the 5’-biotinylated probe MP2-B: 5’-GGT GAA
GGA ATG ATA AGG CT-3’ in the case of M. pneumoniae-PCR. Hybridization signals
were visualized using streptavidin-peroxidase and ECL detection reagents (Amersham,
UK).
Immunohistochemistry
Specimens were transported in 10% phosphate buffered formalin. Subsequently
tissues were, in some cases after decalcification with EDTA, embedded in paraffin and
sectioned at 5 µm. IHC staining was performed according to methods previously
described10. Sections were stained with a mouse anti-C. pneumoniae (TW 183)
monoclonal antibody (DAKO Diagnostics, Denmark). C. pneumoniae-infected HL cells
and a tissue section stained with normal mouse ascites were used as a positive and a
negative control, respectively. Positive and negative controls were run with each batch of
specimens.
26
CORRELATION BETWEEN DETECTION METHODS OF C.PNEUMONIAE
Microimmunofluorescence test
C. pneumoniae (TW 183) IgG antibodies in the sera were determined using a
microimmunofluorescence test (MRL Diagnostics, CA) as described previously11,12. An
IgG antibody titer > 1:16 was considered positive. All serologic tests were performed
blind by the same technician.
Statistical analysis
Correlation between the results of the different assays was analyzed by the
Cohen’s Kappa test.
RESULTS
Atherosclerotic specimens were obtained from 38 males and 7 females (mean
age 63 years, range 41-86). Non-atherosclerotic lesions were obtained from 41 males
and 9 females (mean age 65 years, range 44-79).
Forty-five atherosclerotic tissue specimens and 50 non-atherosclerotic tissue
specimens were available for the PCRs assays. Specimens for IHC assay were obtained
at the same time from all patients, but 3 atherosclerotic specimens and 4 nonatherosclerotic specimens were missed. Blood samples for C. pneumoniae serology
were available from 91 patients (41 in the atherosclerotic group and 50 in the nonatherosclerotic).
C. pneumoniae DNA was detected by PCR in 22% (10/45) of the atherosclerotic
specimens and in 10% (5/50) of non- atherosclerotic specimens. IHC staining was
positive for C. pneumoniae in 60% (25/42) of the atherosclerotic specimens and in 9%
(4/46) of the non-atherosclerotic specimens. The correlation between the detection of C.
pneumoniae by PCR and IHC is shown in table 1. There was a correlation between the
detection of C. pneumoniae by PCR and IHC in the atherosclerotic specimens
(p=0.003). A poor correlation was found between the results obtained by PCR and IHC
in the non-atherosclerotic specimens (p=ns).
The MIF test detected C. pneumoniae IgG antibodies in 88% (36/41) of patients
in the atherosclerotic group and in 96% (48/50) of patients in the non- atherosclerotic
group. Table 2 demonstrates the correlation between C. pneumoniae serology and the
27
CHAPTER 2
results obtained by either PCR and IHC. There was no significant correlation between C.
pneumoniae serology and both PCR and IHC.
Table 1. Correlation between polymerase chain reaction (PCR) and immunohistochemistry (IHC)
in the detection of C. pneumoniae in atherosclerotic and non-atherosclerotic tissue specimens.
atherosclerotic specimens
non-atherosclerotic specimens
IHC negative
IHC negative
PCR negative, n
PCR positive, n
Correlation, Kappa
(p value)
IHC positive
17
0
15
10
IHC positive
38
4
0.35
(0.003)
4
0
0.1
(ns)
Table 2. Correlation between serology (MIF), polymerase chain reaction (PCR) and
immunohistochemistry (IHC) results.
atherosclerotic specimens
PCR negative
MIF negative, n
MIF positive, n
Correlation, Kappa
(p value)
PCR positive
5
26
0
10
IHC negative
IHC positive
2
12
0.1
(ns)
2
22
0.07
(ns)
non-atherosclerotic specimens
PCR negative
MIF negative, n
MIF positive, n
Correlation, Kappa
(p value)
PCR positive
1
44
1
4
IHC negative
IHC positive
2
40
0.04
(ns)
0
4
0.01
(ns)
M. pneumoniae was detected by PCR in one atherosclerotic tissue specimen. All
non-atherosclerotic specimens were M. pneumoniae-negative.
DISCUSSION
This study shows the following features: there was correlation between PCR and
IHC in the detection of C. pneumoniae in atherosclerotic tissues, but a poor correlation
was found in non-atherosclerotic specimens. The MIF test was not correlated with the
results of PCR or IHC. M. pneumoniae was detected in one atherosclerotic specimen. All
non-atherosclerotic specimens were M. pneumoniae-negative.
28
CORRELATION BETWEEN DETECTION METHODS OF C.PNEUMONIAE
Table 3. Correlation between polymerase chain reaction (PCR) and immunohistochemistry (IHC)
in 10 studies in which arterial tissues were investigated.
Correlation,
PCR+ a PCR-b
PCR+
PCRReference
Material
No
Kappa
& IHC+ & IHC- & IHC- & IHC+
(p value)
Campbell et al. Coronary
38
9
18
3
8
0.4 (0.01)
1995
atheromas
Jackson et al.
1997a
Coronary
arteries
38
1
25
5
7
0.05 (0.8)
Kuo et al.
1993b
Coronary
atheromas
30
8
14
5
3
0.45 (0.01)
Shor et al.
1998
Arterial
atheromas
7
2
4
0
1
0.7 (0.05)
Kuo et al.
1997
Peripheral
arteries
17
2
9
0
6
0.3 (0.1)
Juvonen et al.
1997
AAAc
9
5
3
1
0
0.8 (0.02)
Jackson et al.
1997b
Carotid
endarterectomy
16
3
8
0
5
0.4 (0.055)
Ramirez
1996
Coronary
arteries
12
3
5
2
2
0.3 (0.3)
Kuo et al.
1995
Coronary
arteries
49
2
41
1
5
0.3 (0.007)
Davidson et al.
1998
Coronary
arteries
60
12
38
2
8
0.6 (<0.001)
a
+ Positive; b – Negative; c AAA; abdominal aortic aneurysms
Several studies have reported a wide variation in the detection rate of C.
pneumoniae between PCR and IHC5,7. In general, more positive results are obtained
with IHC than PCR. This was also the case in the present study. It has been suggested
that these discrepancies might reflect differences in the sensitivity and specificity of the
two methods1. Focal localization of C. pneumoniae in vascular tissues and the presence
of components that inhibit PCR may influence the results of IHC and PCR7. However,
discrepancy between detection rates of C. pneumoniae by PCR and IHC does not imply
that there is no correlation between these methods. In this study, with regard to the
atherosclerotic tissues there was correlation between the results of IHC and PCR, but no
such correlation was found in non-atherosclerotic specimens. We can not explain why no
correlation was found in non-atherosclerotic tissues. A possible explanation is that after
initial infection, C. pneumoniae does not persist in non-atherosclerotic tissues6. We
analyzed the correlation between PCR and IHC in 10 studies in which these methods
were used to detect C. pneumoniae in arterial tissues6,13-21. Correlation between PCR
29
CHAPTER 2
and IHC results was found in 7 studies (Table 3). Our findings together with previous
report provide evidence that the results of PCR and IHC are correlated.
In the present study no correlation was found between C. pneumoniae serology
and the detection of C. pneumoniae either by PCR and IHC. Controversial reports have
been published on the association between C. pneumoniae serology and the detection
of the pathogen in vascular tissues22. In 7 studies in which C. pneumoniae IgG
antibodies were determined and PCR was used to detect C. pneumoniae in arterial
tissues, correlation between PCR and serological results was found in 3 studies (Table
4)17, 23-28.
Table 4. Correlation between polymerase chain reaction (PCR) and serology (MIF) in 7 studies in
which arterial tissues were investigated.
Correlation,
PCR+ a PCR -b PCR+
PCRReference
Material
No.
Kappa
& MIF+ & MIF- & MIF- & MIF+
(p value)
Kuo et al.
Coronary
29
8
2
5
14
0.2 (0.1)
1993b
atheromas
Maass et al.
1998a
Coronary arteries
158
34
24
0
100
0.1 (0.005)
Blasi et al.
1996
AAA
51
25
9
1
16
0.3 (0.004)
70
21
2
0
47
0.03 (0.3)
47
7
8
0
32
0.07 (0.2)
Maass et al.
1998b
Maass et al.
1997
Carotid
endarterectomy &
restenotic bypass
Carotid
endarterectomy
Blasi et al.
1999
AAA
41
16
14
1
10
0.3 (0.01)
Jantos et al.
1999
Coronary
atheromas
40
2
16
1
21
0.02 (0.7)
a
+ Positive; b - Negative; c AAA; abdominal aortic aneurysms
Large differences in detection prevalence of C. pneumoniae between
atherosclerotic arterial lesions and non-atherosclerotic arterial controls have been
reported, 51% (303 of 597) and 4% (5 of 131), respectively5. This is consistent with the
results of the present study, since C. pneumoniae was more frequently detected in
atherosclerotic tissues. It has been suggested that C. pneumoniae might be less
frequently disseminated to non-atherosclerotic tissues or it does not persist for a long
time in these tissues6.
In a previous study, we detected M. pneumoniae in one of
39 atherectomy
specimens and in two of 64 degenerative heart valves4. In the present study, we
30
CORRELATION BETWEEN DETECTION METHODS OF C.PNEUMONIAE
simultaneously performed C. pneumoniae- and M. pneumoniae-PCR on atherosclerotic
and non-atherosclerotic tissues. One specimen was positive in the M. pneumoniae-PCR,
and 15 specimens were positive in the C. pneumoniae-PCR. These results add a new
evidence for the specificity of the association between C. pneumoniae and vascular
disease.
In conclusion, the results of this study show a good correlation between PCR and
IHC in the detection of C. pneumoniae in atherosclerotic tissues, and support the
specificity of the association between C. pneumoniae and atherosclerotic lesions.
Acknowledgment
We would like to thank M. Kerver, K. Driessen, H. Verbakel and J. Bruinsma for their
technical assistance.
31
CHAPTER 2
REFERENCES
1. Wong YK, Gallagher PJ, ward ME. Chlamydia pneumoniae and atherosclerosis. Heart
1999;81:232-238
2. Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link?
Lancet 1997;350:430-436
3. Taylor-Robinson D, Thomas BJ. Chlamydia pneumoniae in arteries: the facts, their
interpretation, and future studies. J Clin Pathol 1998;51:793-797
4. Maraha B, van der Zee A, Bergmans AMC, Pan ML, Peeters MF, Berg HF, Scheffer GJ,
Kluytmans JAJW. Is Mycoplasma pneumoniae associated with vascular disease? J Clin
Microbiol 2000;38 (2):935-936
5. Gibbs RGJ, Carey N, Davies AH. Chlamydia pneumoniae and vascular disease. Br J Surg
1998; 85:1191-1197
6. Jackson LA, Campbell LA, Schmidt RA, Kuo CC, Cappuccio AL, Lee MJ, Grayston JT.
Specificity of detection of Chlamydia pneumoniae in cardiovascular atheroma. Evaluation of
the innocent bystander hypothesis. Am J Pathol 1997;150:1785-1790
7. Kuo CC, Campbell LA. Detection of Chlamydia pneumoniae in arterial tissues. J Infect Dis
2000;181 (suppl 3):S432-S436
8. Gaydos CA, Quinn TCJ, Eiden J. Identification of Chlamydia pneumoniae by DNA
amplification of the 16S rRNA gene. J Clin Microbiol 1992;30:796-800
9. Ieven M, Ursi D, Van Bever H, Quint W, Niesters HGM, Goossens H. Detection of
Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in
acute respiratory tract infections in pediatric patients. J Infect Dis 1996;173:1445-1452
10. Kuo CC, Gown AM, Benditt EP, Grayston JT. Detection of Chlamydia pneumoniae in aortic
lesions of atherosclerosis by immunocytochemical stain. Arterioscler Thromb 1993;13:15011504
11. Freidank HM, Vögele H, Eckert K. Evaluation of new commercial microimmunofluorescence
test for detection of antibodies to Chlamydia pneumoniae, Chlamydia trachomatis, and
Chlamydia psittaci. Eur J Clin Microbiol Infect Dis 1997;16:685-688
12. Peeling RW, Wang SP, Grayston JT, Blasi F, Boman J, Clad A, Freidank H, Gaydos CA,
Gnarpe J, Hagiwara T, Jones RB, Orfila J, Persson K, Puolakkainen, Saikku P, Schachter J.
Chlamydia pneumoniae serology: interlaboratory variation in microimmunofluoresence assay
results. J Infect Dis 2000;181 (suppl 3):S426-S9
13. Campbell LA, O’Brien ER, Cappuccio AL, Kuo CC, Wang SP, Stewart D, Patton DL,
Cummings PK, Grayston JT. Detection of Chlamydia pneumoniae TWAR in Human coronary
atherectomy tissues. J Infect Dis 1995;172:585-588
14. Davidson M, Kuo CC, Middaugh JP, Campbell LA, Wang SP, Newman III WP, Finley J,
Grayston JT. Confirmed previous infection with Chlamydia pneumoniae (TWAR) and its
presence in early coronary atherosclerosis. Circulation 1998;98:628-633
15. Jackson LA, Campbell LA, Kuo CC, Rodriguez DI, Lee MJ, Grayston JT. Isolation of
Chlamydia pneumoniae from a carotid endarterectomy specimen. J Infect Dis 1997;176:292295
32
CORRELATION BETWEEN DETECTION METHODS OF C.PNEUMONIAE
16. Juvonen J, Juvonen T, Laurila A, Alakarppa H, Lounatmaa A, Surcel HM, Leinonen M,
Kairaluoma MI, Saikku P. Demonstration of Chlamydia pneumoniae in the walls of abdominal
aortic aneurysms. J Vasc Surg 1997;25:499-505
17. Kuo CC, Shor A, Campbell LA, Fukushi H, Patton DL, Grayston JT. Demonstration of
Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J Infect Dis
1993;167:841-849
18. Kuo CC, Grayston JT, Campbell LA, Goo YA, Wisseler RW, Benditt EP. Chlamydia
pneumoniae (TWAR) in coronary arteries of young adults (15-34 years old). Proc Natl Acad
Sci USA 1995;92:6911-6914
19. Kuo CC, Coulson AS, Campbell LA, Cappuccio AL, Lawrence RD, Wang SP, Grayston JT.
Detection of Chlamydia pneumoniae in atherosclerotic plaques in the walls of arteries of
lower extremities from patients undergoing bypass operation for arterial obstruction. J Vasc
Surg 1997;26:29-31
20. Ramirez JA. Isolation of Chlamydia pneumoniae from the coronary artery of a patient with
coronary atherosclerosis. Ann Intern Med 1996;125:979-82
21. Shor A, Philips JI, Ong G, Thomas BJ, Taylor-Robinson D. Chlamydia pneumoniae in
atheroma: consideration of criteria for causality. J Clin Pathol 1998;51:812-817
22. Bartels C, Maass M, Bein G, Brill N, Matthias JF, Leyh R, Sievers HH. Association of
serology with the endovascular presence of Chlamydia pneumoniae and Cytomegalovirus in
coronary artery and vein graft disease. Circulation 2000;101:137-141
23. Blasi F, Denti F, Erba M, Cosentini R, Raccanelli R, Rinaldi A, Fagetti L, Esposito G, Ruberti
U, Allegra L. Detection of Chlamydia pneumoniae but not Helicobacter pylori in
atherosclerotic plaques of aortic aneurysms. J Clin Microbiol 1996;34:2766-2769
24. Blasi F, Boman J, Esposito G, Melissano G, Chiesa R, Cosentini R, Tarsia P, Tshomba Y,
Betti M, Alessi M, Morelli N, Allegra L. Chlamydia pneumoniae DNA detection in peripheral
blood mononuclear cells is predictive of vascular infection. J Infect Dis 1999;180:2074-2076
25. Jantos CA, Nesseler A, Waas W, Baumgärtner W, Tillmanns H, Haberbosch W. Low
prevalence of Chlamydia pneumoniae in atherectomy specimens from patients with coronary
heart disease. Clin Infect Dis 1999;28:988-992
26. Maass M, Krause E, Engel PM, Kruger S. Endovascular presence of Chlamydia pneumoniae
in patients with hemodynamically effective carotid artery stenosis. Angiology 1997;48:699706
27. Maass M, Gieffers J, Krause E, Engel PM, Bartels C, Solbach W. Poor correlation between
microimmunofluorescence serology and polymerase chain reaction for detection of
Chlamydia pneumoniae infection in coronary artery disease patients. Med Microbiol Immunol
1998;187:103-106
28. Maass M, Bartels C, Engel PM, Mamat M, Siervers HH. Endovascular presence of viable
Chlamydia pneumoniae is a common phenomenon in coronary artery disease. J Am Coll
Cardiol 1998;31:827-832
33
CHAPTER
3
EXTRACTION OF CHLAMYDIA PNEUMONIAE-DNA FROM VASCULAR
TISSUE FOR USE IN PCR: AN EVALUATION OF FOUR PROCEDURES
HF Berg, B Maraha, AMC Bergmans, A van der Zee, JAJW Kluytmans, MF Peeters
Clinical Microbiology and Infection 2003;9:135-139
CHAPTER 3
SUMMARY
The objective of this study was to compare four procedures for Chlamydia pneumoniaeDNA extraction from vascular tissue. NucliSensTMKit, QIAamp® tissue DNA MiniKit,
buffer-saturated phenol, and Geneclean II® Kit, were evaluated, based on the yield of
recovered DNA using PCR to detect Chlamydia pneumoniae in vascular tissue. The
QIAamp® tissue procedure had the highest detection level (0.004 IFU / sample). All
methods, except NucliSens (70 minutes), had a short handling time (30-40 minutes).
Costs varied from 0.5 to 3.2 euro.
36
EVALUATION OF FOUR DNA-EXTRACTION METHODS
INTRODUCTION
Chlamydia (C.) pneumoniae has been associated with atherosclerosis. First of all,
seroepidemiological studies suggested this association1,2. Two recent studies, however,
have failed to demonstrate any such connection3,4. Subsequently, further evidence has
been provided by the detection of C. pneumoniae in atherosclerotic tissue by
polymerase chain reaction (PCR), immunocytochemistry5-7, and also by isolation in
culture. Whether C. pneumoniae plays a role in the pathogenesis of atherosclerosis is
still not known. Although serological assays are considered the reference diagnostic
method for C. pneumoniae infections, PCR is potentially an important tool for further
studies in this field. C. pneumoniae-PCR is however not yet standardised. Several PCR
assays have been used to detect C. pneumoniae in vascular tissues. However, a
considerable variation in the detection rate of C. pneumoniae, ranging from 0% to 100%,
has been reported by different investigators5-10. This phenomenon can be explained by
the differences between the populations studied, by sampling error, but also by the
difference between the PCR techniques applied. The potential for differences in
performance between PCR techniques is well known. For example, the sensitivity of
PCR for the detection of Mycobacterium tuberculosis in samples containing low numbers
of micro-organisms, varied among seven laboratories from 2 – 90%11. A multicenter
study showed major inter-laboratory differences in the detection rate of C. pneumoniae
in endarterectomy specimens12. Therefore, it is important to arrive at a more
standardised procedure including DNA extraction from specimens. In order to detect C.
pneumoniae by PCR, efficient release of C. pneumoniae-DNA from vascular tissue and
adequate removal of PCR inhibitors (presence of lipids and calcification) are essential13.
The purpose of this study is to compare four procedures of C. pneumoniae-DNA
extraction from vascular tissue.
MATERIAL & METHODS
Vascular samples were obtained from 30 patients during cardiac surgery (CABG).
During this procedure a punch through the aortic wall is routinely taken. This punch was
stored at 4ºC in 200 µl of lysis buffer (1M Tris pH 7.0, 0.5 mM EDTA, 5M NaCl, 1% SDS,
20 mg/ml proteinase K) for a maximum of 24 hours. Subsequently, the sample was lysed
37
CHAPTER 3
by adding 20 µl proteinase-K (20mg/ml, Qiagen) and incubating overnight at 56ºC. After
cell lysis a homogeneous solution was made by pooling all 30 lysates. Seven 900 µl
portions of the homogenate were inoculated with decreasing concentrations of inclusionforming units (IFU) of C. pneumoniae strain TW 183. Portion number eight, containing
AE buffer (QIAamp, Qiagen, Germany), was used as a negative control.
Two hundred and five µl of each portion (corresponding to the concentrations of
the dilution series (ten- and fivefold): 10,000, 1000, 100, 10, and 2, 0.4, 0.08 IFU per 205
µl; per PCR this is: 500, 50, 5, 0.5, 0.1, 0.02 and 0.004 IFU) was subjected to each of
four methods of DNA extraction:
Method I. Using NucliSensTM (Organon Teknika, Boxtel, NL), based on the method of
Boom et al.14, according to manufacturer’s instructions. This technique is based on
the mechanism whereby DNA binds to glass particles (silica) in a high
concentration
of
chaotropic
salt,
while
contaminants
such
as
proteins,
carbohydrates, and ions do not. A wash procedure is repeated three times to
remove all contaminants (with wash buffer, or 70% ethanol or acetone). DNA is
eluted
from
the
silica
by
resuspension
of
the
silica
complexes
in
NucliSens™elution buffer.
Method II. Using QIAamp® DNA MiniKit ( Qiagen, Hilden, Germany), according to the
QIAamp tissue protocol of the manufacturer’s instructions. This method uses a
QIAamp spin column to which DNA binds in the presence of buffer AL and ethanol.
Two wash steps, in which AW1 buffer and AW2 buffer succeed each other, are
performed to remove contaminants. AE buffer is finally used for elution of the DNA
from the spin column.
Method III. Using buffer-saturated phenol (Life Technologies, Inc. US California). This
method is home-made, based on "classical" phenol extraction. In this method 200
µl of buffer-saturated phenol (pH 7.5 – 7.8) was added to 205 µl of sample in a 1.5ml screw-cap plastic tube, vortexed for 1 min and centrifuged for 5 min at 20,000 x
g. The aqueous supernatant was transferred to another tube which also contained
200 µl of buffer-saturated phenol. After homogenizing and centrifuging (5 min at
20,000 x g), the aqueous supernatant was transferred to a 1.5-ml screw-cap
plastic tube containing 20 µl 3 M NaAc (pH 5,2) and vortexed. 440 µl of ice-cold
absolute ethanol was added, the mixture homogenized and incubated at -20°C for
15 min. The sample was centrifuged (15 min, 20,000 x g) to pellet the DNA
products. The supernatant was removed, and the pellet resuspended in 250 µl of
38
EVALUATION OF FOUR DNA-EXTRACTION METHODS
70% ethanol, vortexed and centrifuged for 5 min (20,000 x g). The supernatant
was removed again, and a quick centrifuge spin was done, so that the remaining
ethanol could be removed. The pellet was air dried in a half-open tube and
suspended in 100 µl of AE buffer by vortexing.
Method IV. Using the Geneclean II® Kit (Qbiogene, Illkirch Cedex, France), according to
manufacturer’s instructions. Like method I, this method is also based on the
mechanism that DNA binds to glass particles in a high concentration of chaotropic
salt. Here the DNA binds to Glassmilk®. The Glassmilk®/DNA pellet is washed
once with New Wash®.
All DNA elutions (in 100 µl of AE buffer, Qiagen) were resuspended at 80°C for 5 min,
which completed the DNA extraction.
An identical PCR assay was performed on 5 µl of each sample to detect C. pneumoniaeDNA, irrespective of extraction method. All amplification steps, assay conditions, signals,
visualisation steps and hybridisation procedures were identical.
Polymerase chain reaction
Primers CpnA: 5’-TGA CAA CTG TAG AAA TAC AGC-3’ and CpnB: 5’-CGC CTC TCT
CCT ATA AAT-3’ were used in a PCR based on the 16S rRNA gene sequence as
described by Gaydos et al.15.
The PCR reaction mixture contained 30 pmol of each primer, 3 mM MgCl2, 200
µM dNTPs (dTTP is replaced by dUTP), 2.5 units of AmpliTaq Gold DNA polymerase
(Perkin Elmer Cetus, Norwalk, Conn.), and 10 x PCR buffer II (Perkin Elmer), and 1.25
µl of internal control.
An internal control was added in each reaction to be able to detect inhibition of
the PCR reaction and prevent false-negative PCR results. The internal control template
DNA consisted of a PCR product of an unknown fragment of E. coli DNA, that yields a
150bp-PCR product in combination with primer PINTK (5’- (ACTG x 4)-AC-3’).
The PCR amplification was performed as follows: after addition of 5 µl of
template DNA in a final volume of 25 µl PCR reaction mixture, samples were subjected
to the following PCR program: 10 min at 96ºC, followed by 40 cycles of 30s at 95ºC, 30s
at 55ºC, and 1 min at 72ºC. A final step of 10 min at 72ºC completed the PCR in a
thermocycler (9600, Perkin Elmer). A negative PCR mix control and a negative sample
processing control were included in each PCR run with every 5 samples to detect falsepositive results.
39
CHAPTER 3
For final product detection, amplification products were examined by agarose gel
electrophoresis and dot-blot hybridization as follows: eight µl of each PCR product was
analysed by agarose gel-electrophoresis on 2% agarose gels in TBE buffer containing
ethidium-bromide. PCR products were visualized on UV transillumination and
photographed. If the 450 base-pair (bp) Chlamydia-derived band was visible (with or
without the 150 bp band), the sample was considered positive. If only the 150 bp band of
the internal control was visible, the sample was considered negative. If no bands were
visible, the PCR was considered inhibited, and the sample was repurified and retested
by PCR.
Dot-blot hybridization: hybridization of 5 µl of the PCR products was performed
using
a
5’-biotinylated
C.
ACACACGTGCTACAATGGTT-3’.
streptavidin-peroxidase
pneumoniae
specific
probe,
Hybridization
signals
were
(Boehringer
Mannheim)
and
ECL
Cpneu-B:
visualized
detection
5’using
reagents
(Amersham).
To minimise the risk of contamination, sample preparation, PCR amplification,
and analysis of the PCR product were performed in separate rooms.
Analysis. comparison of the four procedures was done using gel electrophoresis
and dot-blot hybridization. Each procedure was also compared for overall-time
consumption and hands-on-time per sample. Hands-on-time was defined as time
needed by the technician working with this procedure, overall-time as the total of time
including centrifuging, incubation, etc. Finally, the average costs per sample were
estimated in euro's, calculated by the price of the commercial kit and material (e.g.
ethanol), excluding the use of materials such as plastic tubes, divided by the number of
samples.
RESULTS
Figure 1 shows the results of C. pneumoniae detection for each method. The detection
levels ranged from 0.004 IFU per sample for QIAamp® to 0.1 IFU per sample for Phenolextraction and NucliSens, and 0.5 IFU per sample for Geneclean II (table 1).
Time consumption was in favour of Geneclean II, with a hands-on-time of thirty
minutes, and an overall-time of sixty minutes and therefore the least labour-intensive
(table 1). Costs per sample of various methods shows that buffer-saturated phenol was
the cheapest method with an average of 0.5 euro per sample (table 1).
40
EVALUATION OF FOUR DNA-EXTRACTION METHODS
NucliSens
QIAamp
*Æ
*Æ
Phenol
Genclean II
Figure 1. Agarose gel electrophoresis of
PCR products (Chlamydia pneumoniae)
using four DNA extraction methods.
* Internal control bands
DISCUSSION
In this study, we compared four different DNA extraction methods. To create realistic
study material, a homogenate solution was prepared from aorta tissue samples
inoculated with IFU’s from C. pneumoniae. The QIAamp® DNA MiniKit extraction method
detected the lowest amount of IFU’s by far. Also it is a rapid and easy-to-perform
procedure. The associated costs are a disadvantage.
Several factors influence the ability of PCR to detect C. pneumoniae, including
sample preparation, DNA extraction, amplification assays, and visualisation procedures.
Standardisation of these factors was therefore approached in the present study.
Because C. pneumoniae is an intracellular pathogen, vascular tissue was treated
with proteinase-K to produce tissue cell lysis and release C. pneumoniae-DNA, if
present. The aorta samples used in the present study were macroscopically nonatherosclerotic, and the expected positivity rate was low16. Since the amount of C.
41
CHAPTER 3
pneumoniae in the study materials was unknown, a homogeneous pool of all 30 lysates
was made and inoculated with decreasing concentrations of IFU's to be able to compare
DNA extraction methods. It should be mentioned that the concentration of C.
pneumoniae in the dilution series is very similar, though it is impossible to achieve
identical concentrations. Moreover, it is not known whether the ability of the four
procedures to extract C. pneumoniae-DNA from spiked materials is the same as their
ability to extract DNA from patient materials12.
A multicenter study12 demonstrated that the sensitivity of a PCR assay does not
necessarily correspond with the ability to detect C. pneumoniae in patient material
without a logical explanation. In the present study we performed one PCR assay, and
because there is no reference assay available, one should view the results of the
present study in the light of the lack of a reference PCR assay.
Taking the limitations of the present study into consideration, we can conclude
that QIAamp is a useful and sensitive DNA-extraction method, but further effort to
optimise and standardise DNA-extraction methods is needed.
ACKNOWLEDGEMENT
We thank M. E. Kerver and J. M. Verbakel for their technical assistance.
42
EVALUATION OF FOUR DNA-EXTRACTION METHODS
REFERENCES
1. Gupta S, Camm AJ. Chlamydia pneumoniae and coronary heart disease. Br Med J 1997;
314:1778-1779
2. Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link?
Lancet 1997; 350:430-436
3. Ridker PM, Kundsin RB, Stampfer MJ, Poulin S, Hennekens CH. Prospective study of
Chlamydia pneumoniae IgG seropositivity and risks of future myocardial infarction. Circulation
1999;99:1161-1164
4. Danesh J, Wong Y, Ward M, Muir J. Chronic infection with Helicobacter pylori, Chlamydia
pneumoniae, or cytomegalovirus: population based study of coronary heart disease. Heart
1999;81:245-247
5. Kuo C-C, Grayston JT, Campbell LA, Goo YA, Wissler RW, Benditt EP. Chlamydia
pneumoniae (TWAR) in coronary arteries of young adults (15-34 years old). Proc Nathl Acad
Sci USA 1995;92:6911-6914
6. Jackson LA, Campbell LA, Schmidt RA, Kuo CC, Cappuccio AL, Lee MJ, Grayston JT.
Specificity of detection of Chlamydia pneumoniae in cardiovascular atheroma: evaluation of
the innocent bystander hypothesis. Am J Pathol 1997;150:1785-90
7. Campbell LA, O’Brien ER, Cappuccio AL, Kuo CC, Wang SP, Stewart D, Patton DL,
Cummings PK, Grayston JT. Detection of Chlamydia pneumoniae TWAR in human coronary
atherectomy tissues. J Infect Dis 1995;172:585-588
8. Andreasen JJ, Farholt S, Jensen JS. Failure to detect Chlamydia pneumoniae in calcific and
degenerative arteriosclerotic aortic valves excised during open heart surgery. APMIS
1998;106:717-720
9. Juvonen J, Juvonen T, Laurila A, Alakarppa H, Lounatmaa A, Surcel HM, Leinonen M,
Kairaluoma MI, Saikku P. Demonstration of Chlamydia pneumoniae in the walls of abdominal
aortic aneurysms. J Vasc Surg 1997;25:499-505
10. Lindtholdt JS, Ostergard L, Henneberg EW, Fasting H, Anderson P. Failure to demonstrate
Chlamydia pneumoniae in symptomatic abdominal aortic aneurysms by a nested polymerase
chain reaction (PCR). Eur J Vasc Endovasc Surg 1998;15:161-4
11. Noordhoek GT, Kolk AH, Bjune G, Catty D, Dale JW, Fine PEM, Godfrey-Faussett P, Cho
SN, Shinnick T, Svenson SB, Wilson S, Embden JDA. Sensitivity and specificity of PCR for
detection of Mycobacterium tuberculosis: a blind comparison study among seven
laboratories. J Clin Microbiol 1994 ;32:277-284
12. Apfalter P, Blasi F, Boman J, Gaydos CA, Kundi M, Maass M, Makristathis A, Meijer A,
Nadrchal R, Persson K, Rotter ML, Tong CY, Stanek G, Hirschl AM. Multicenter comparison
trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in
endarterectomy specimens. J Clin Microbiol 2001; 39:519-524
13. Boman J, Gaydos CA, Quinn TC. Molecular diagnosis of Chlamydia pneumoniae infection. J
Clin Microbiol 1999; 37:3791-3799
14. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J.
Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990 ;28:495-503
43
CHAPTER 3
15. Gaydos CA, Quinn TC, Eiden JJ. Identification of Chlamydia pneumoniae by DNA
amplification of the 16S rRNA gene. J Clin Microbiol 1992;30:796-800
16. Maraha B, Berg H, Scheffer GJ, van der Zee A, Bergmans A, Misere J, Kluytmans J, Peeters
M. Correlation between detection methods of Chlamydia pneumoniae in atherosclerotic and
non-atherosclerotic tissues. Diagn Microbiol Infect Dis 2001;39:139-143
44
CHAPTER
4
EFFECT OF CLARITHROMYCIN TREATMENT ON CHLAMYDIA
PNEUMONIAE IN VASCULAR TISSUE OF PATIENTS WITH CORONARY
ARTERY DISEASE: A RANDOMISED, DOUBLE-BLIND, PLACEBOCONTROLLED TRIAL
HF Berg, B Maraha, A van der Zee, SK Gielis, PJM Roholl, GJ Scheffer, MF Peeters,
JAJW Kluytmans
Submitted
CHAPTER 4
SUMMARY
Small clinical trials have indicated that antibiotic treatment of Chlamydia (C.)
pneumoniae has clinical beneficial effects in patients with coronary artery disease
(CAD). It has not been demonstrated if antibiotic treatment eradicates C. pneumoniae
from vascular tissue. The aim of this study was to assess the effect of clarithromycin on
the presence of C. pneumoniae in vascular tissue of patients with CAD.
Patients with CAD, waiting for coronary artery bypass graft surgery were enrolled into a
randomised, double-blind, placebo-controlled trial. Patients were randomly assigned to
receive clarithromycin 500 mg or placebo once daily, from the day of inclusion till
surgery. During surgery vascular specimens were obtained. The presence of C.
pneumoniae in vascular specimens was examined by immunohistochemistry (IHC) and
two polymerase chain reaction (PCR) assays. Baseline Chlamydia IgG titers and titers 8
weeks after surgery were determined using an enzyme immunoassay.
A total of 76 patients were included and 180 vascular specimens were obtained, 80
specimens from the clarithromycin group and 100 specimens from the placebo group. 35
patients received clarithromycin (mean= 27 days, SD =12.2) and 41 placebo (mean=27
days, SD=13.9). IHC detected C. pneumoniae major outer membrane protein antigen in
73.8% of specimens in the clarithromycin group vs. 77.0% in the placebo group (p=ns).
Chlamydia lipopolysaccharide antigen was found in one placebo specimen. C.
pneumoniae DNA was not detected by PCR in any specimen. Baseline Chlamydia IgG
titers were equally distributed in both groups and were not significantly different after
treatment.
Clarithromycin treatment of patients with CAD has neither an effect on the presence of
C. pneumoniae in vascular tissue nor on Chlamydia IgG titers.
46
EFFECT OF CLARITHROMYCIN ON CHLAMYDIA PNEUMONIAE IN VASCULAR TISSUE
INTRODUCTION
Many risk factors for atherosclerosis are well defined. However, atherogenesis is not
fully understood and infectious pathogens, particularly Chlamydia (C.) pneumoniae,
have been considered as potential risk factors for atherosclerosis1. It has been supposed
that, during respiratory tract infection, C. pneumoniae reaches vascular tissue via
infected leukocytes. In vascular tissue, C. pneumoniae can infect atheroma-associated
cells and induce inflammatory cytokines production and smooth muscle cells
proliferation2. C. pneumoniae may also cause endothelial dysfunction and promote the
secretion of matrix-degrading metalloproteinases that destabilize the atherosclerotic
plaque2,3. Chlamydial lipopolysaccharide (LPS) and chlamydial heat shock protein 60 kd
may contribute to atherogenesis by promoting foam cells formation, lipoprotein oxidation
and proinflammatory activation2.
Some seroepidemiologic studies have found an association between C.
pneumoniae and coronary artery disease (CAD). Prospective serologic studies,
however, failed to demonstrate any association4. Further indications that C. pneumoniae
might play a role in atherogenesis came from studies that identified C. pneumoniae in
vascular tissue by polymerase chain reaction (PCR), immunohistochemical staining
(IHC), electron microscopy, and culture1. However, the results of these studies are
inconsistent and a huge variation in the detection rate has been reported1,5.
The results of pilot clinical trials that showed beneficial effects from antibiotic
treatment encouraged many groups to further investigate the effect of antibiotic
treatment on secondary prevention of cardiovascular events6-8. These studies are based
on the hypothesis that antibiotic treatment of C. pneumoniae in CAD patients will
stabilize atheromas and decrease cardiovascular events. However, it has not been
studied whether antibiotic treatment eradicates C. pneumoniae from vascular tissue of
CAD patients. We initiated in 1999 a placebo-controlled, double-blind, randomised
clinical trial to investigate the effect of clarithromycin treatment on the presence of C.
pneumoniae in vascular tissue, and on circulating Chlamydia IgG antibodies in patients
with CAD. Two PCR assays and IHC staining were used to assess the eradication of C.
pneumoniae from vascular tissue.
47
CHAPTER 4
PATIENTS AND METHODS
Study population
Between July 1999 and July 2001, patients with documented CAD and scheduled for
coronary artery bypass graft (CABG) surgery were invited to participate in the study.
Inclusion was carried out during attendance at the pre-operative outpatient clinic at the
department of thoracic surgery of the Amphia Hospital. Exclusion criteria included: (I)
concomitant administration of terfenadine, rifabutin or cisapride; (II) Antibiotic therapy
with a macrolide, tetracycline, or quinolone within three months prior to inclusion or
during the study period; (III) Renal failure (serum creatinine rates above 150 µmol/l); (IV)
elevated liver function (alanine-aminotransferase > 55 U/l, aspartate-aminotransferase >
45 U/l, total bilirubin > 27 µmol/l, or alkaline phosphatase > 180 U/l); (V) female patients
capable of child-bearing but not taking adequate birth control precautions.
After giving informed consent, patients were randomised in a double-blind,
placebo controlled trial. Patients received, from the day of inclusion till the day of
surgery, a daily dose of clarithromycin 500 mg slow release (SR) or a placebo tablet
(Clarithromycin SR and matching placebo tablets were obtained from Abbott
laboratories, Abbott Laboratories Ltd, Queenborough, Kent, England ME11 5EL).
An independent pharmacist dispensed either clarithromycin or placebo tablets
according to a computer-generated randomisation table, which stratified in groups of 10.
The researcher responsible for seeing the patients allocated the next available number
on entry into the trial, and provided the patient the corresponding tablets. The code was
revealed to the researcher once recruitment, data collection, and laboratory analysis
were complete. The local Medical Ethics Committee approved the study.
Clinical specimens
During CABG surgery specimens were obtained from coronary atheromas, obstructed
old coronary grafts, mammary artery, saphenal vein, when possible. All specimens were
divided into two portions, one for IHC and one for PCR. IHC samples were routinely fixed
in 10% buffered formalin until further research. PCR samples were transported at 4ºC in
200 µl of lysis buffer (1M Tris pH 7.0, 0.5 mM EDTA, 5M NaCl, 1% SDS, 20 mg/ml
proteinase K), and processed within 24 hours.
48
EFFECT OF CLARITHROMYCIN ON CHLAMYDIA PNEUMONIAE IN VASCULAR TISSUE
From each patient 10 ml blood was obtained on the day of inclusion and 8 weeks after
surgery. Blood was stored at 4ºC immediately after collection, and centrifuged within two
hours. Serum was then stored at -20ºC pending further testing.
Laboratory methods
Serology.
Chlamydia IgG antibody titers were determined by a recombinant enzyme
immunoassay (rELISA, Medac GmbH, Hamburg, Germany) according to the
manufacturer’s instructions. This rELISA uses a recombinant Chlamydia-specific LPS
fragment as antigen. IgG titer of > 1:100 was considered positive.
Immunohistochemistry.
Cross-sections of each paraffin wax embedded vascular
specimen were stained with hematoxilin-eosin stain. In each cross-section, the lumen
area, the circumference of the internal elastic lamina and the area encompassed by it,
were evaluated. Antigens were detected in 4 µm sections by IHC as described by Meijer
et al.9. In the IHC, two monoclonal antibodies were used. The species specific
monoclonal antibody RR-402 against C. pneumoniae major outer membrane protein
(MOMP) (Washington Research Foundation, Seattle, Washington, USA)10, and the
Chlamydia genus specific anti-LPS monoclonal antibody 16.3B6 (produced by the
National Institute of Public Health and the Environment, Bilthoven, The Netherlands).
HEp2 cells (CCL23; American Type Culture Collection) infected with C. pneumoniae
strain TW-183 were used as positive control, and mock-infected HEp2 cells were used
as negative control.
The specimens were evaluated microscopically by one experienced technician.
Specimens were considered positive for C. pneumoniae antigen when a clear dot-likecell associated staining was observed5.
Polymerase chain reaction
Specimens processing.
Within 24 hours after surgery, DNA was extracted from
clinical specimens by the QIAamp DNA mini kit (Qiagen Inc., Valencia, Calif.) according
to the manufacturer’s instructions. A control was included with every four clinical
specimens in the extraction procedure.
Real-time PCR.
A real-time PCR assay specific for C. pneumoniae and designed
to the VD4 variable domain of the ompA gene was performed. Oligonucleotide primers
included VD4 forward primer (5'-TCC GCA TTG CTC AGC C-3'), VD4 reversed primer
49
CHAPTER 4
(5'-AAA CAA TTT GCA TGA AGT CTG AGA A-3'), and a VD4 probe (5'-FAM-TAA ACT
TAA CTG CAT GGA ACC CTT CTT TAC TAG G-TAMRA)11.
To be able to monitor possible inhibition of PCR in the clinical specimens a
universal internal control was used. This internal control sample consisted of a wholevirus preparation of phocid herpesvirus (PhHV-1),12 which was added to the original
clinical sample at a final concentration of approximately 5,000 to 10,000 DNA copies per
ml. Primers PhHV-F1 (5'-GGG CGA ATC ACA GAT TGA ATC-3'), PhHV-R1 (5'-GCG
GTT CCA AAC GTA CCA A-3') and probe (5'-VIC-TTT TTT ATG TGT CCG CCA CCA
TCT GGA TC-TAMRA-3') were used to amplify PhHV1, which in uninhibited samples
had a cycle threshold value of approximately 30. Amplification was carried out with both
C. pneumoniae and PhHV1 specific primers and probes in a multiplex PCR.
Reactions were prepared with 96-well MicroAmp optical plate (Applied
Biosystems) by addition of 5 µl of extracted DNA to 45 µl of PCR mixture containing 1x
TaqMan universal PCR master mix (Applied Biosystems), 600nM VD4 forward primer,
300nM VD4 reversed primer, and 150 nM FAM fluorescent C. pneumoniae specific
probe, 5 µl PhHV1 (whole-virus), 400 nM PhHV-F1 forward primer, 400 nM PhHV-R1
reversed primer, and 150 nM VIC-labeled PhHV-1 specific probe. The 96-well plate was
centrifuged for 1 min at 1,000 xg at room temperature in a swing-out rotor to remove
small air bubbles in the reaction vessels. Amplification and detection were performed
with an ABI Prism 7900HT sequence detection system (Applied Biosystems) by using the
manufacturer's standard protocols. The PCR cycling program consisted of 2 min at
50°C, 10 min at 95°C, and 50 cycles of 15s at 95°C and 1 min at 60°C. Each run
contained: (I) negative controls (one for every four extracted DNA samples); (II) positive
control series containing a known amount of inclusion forming units (IFU) of C.
pneumoniae (5, 2, and 1 IFU); (III) a negative mix control. A specimen was considered
positive for C. pneumoniae if the fluorescence was above the threshold limit. Specimens
were considered negative for C. pneumoniae if the internal control was positive with a
cycle threshold value of < 35.
Industry-developed research-use-only LCx C. pneumoniae PCR (RUO-PCR).
The presence of C. pneumoniae DNA in specimens was also examined by an Industrydeveloped LCx C. pneumoniae RUO-PCR (Diagnostics Division, Abbott Laboratories,
Abbott Park, Chicago, Illinois, USA). The RUO-PCR assay was performed at Abbott
Laboratories by Abbott personnel as described earlier13. Briefly, an activation mixture
was prepared by mixing equal volumes of LCx Activation Reagent II and LCx C.
50
EFFECT OF CLARITHROMYCIN ON CHLAMYDIA PNEUMONIAE IN VASCULAR TISSUE
pneumoniae Oligo Mix. 40 µl of the freshly prepared activation mixture and 30 µl of the
purified DNA samples were subsequently added to the appropriate LCx amplification
vial. The total reaction volume was 200 µl. Amplification was carried out with a 480
thermocycler (Perkin-Elmer, Norwalk, Conn.) under the following conditions: 97°C for 2
min; 40 cycles of 97°C for 30 s, 59°C for 30 s, and 72°C for 30 s; and finally, 1 cycle of
97°C for 5 min and 12°C for 5 min. PCR products were detected with the LCx Analyzer.
Samples yielding a rate over 100 cps per second were considered C. pneumoniae
positive. This cutoff was determined by testing titrated C. pneumoniae isolates and
uninfected HEp-2 cell DNA multiple times13.
Specimens were coded and all detection experiments were performed blind. The
code was revealed when the study was completed.
Statistical analysis
All baseline characteristics were analyzed using a χ2-test or a Student’s t test when
appropriate. A value of P<0.05 was considered statistically significant. The SPSS 11.0
statistical software package was used for all calculations.
80 total eligible patients
80 patients randomized
38 clarithromycin
42 placebo
3 excluded from analysis
because of concomitant
use of other antibiotics
during study period
1 excluded from analysis
because of concomitant use
of other antibiotics during
study period
35 analyzed
41 analyzed
Figure 1. Trial Profile.
51
CHAPTER 4
RESULTS
Figure 1 shows that a total of 80 patients with CAD, waiting for CABG surgery, were
enrolled in the study. Four patients who during the study period concomitantly used
other antibiotics were excluded from this treatment analysis due to possible additive
effects. Thirty-five patients were randomly assigned to receive clarithromycin and 41 to
receive placebo. Table 1 shows that the baseline patient characteristics are well
balanced between the two treatment groups. The mean number of treatment-days, for
both groups, was 27 as indicated by the number of tablets used.
Tabel 1. Baseline patient characteristicsa.
Characteristics
Male
Age, mean
Weight, mean
Length, mean
NYHA-score, mean
Treatment-days, mean
Current smoker
Smoker in past
Medical history
COPD
Diabetes Mellitus - Type I
- Type II
Hypercholesterolaemia
Malignancy
CVA or TIA
Pectoral angina
Myocardal infarction
Valvular insufficience
Hypertension
Earlier vascular surgery
Family medical history
Cardiovascular disease
Diabetes mellitus
Medication
Statins
Anti-hypertensive drugs
Clarithromycin,
n = 35
32 (91.4)
65 (8.6)
84 (19.4)
170 (20.2)
3 (0.9)
27 (12.2)
5 (14.3)
27 (77.1)
Placebo,
n = 41
35 (85.3)
65 (9.3)
81 (12.1)
174 (9.9)
3 (0.7)
27 (13.9)
4 (9.8)
31 (75.6)
6 (17.1)
2 (5.7)
4 (11.4)
20 (57.1)
3 (8.6)
2 (5.7)
33 (94.3)
20 (57.1)
3 (8.6)
18 (51.4)
10 (28.6)
3 (7.3)
1 (2.4)
8 (19.5)
27 (65.9)
1 (2.4)
6 (14.6)
40 (97.6)
23 (56.1)
1 (2.4)
25 (61.0)
15 (36.6)
29 (82.9)
7 (20.0)
31 (75.6)
6 (14.6)
21 (60.0)
35 (100)
27 (65.9)
41 (100)
Values are means (SD) or numbers (%);
a
Baseline patient characteristics are not significantly different between the treatment groups.
During CABG surgery, 180 vascular specimens were obtained, including coronary
atheromas (n=31), obstructed old CABG specimens (n=12), mammary artery specimens
(n=66), and saphenal vein specimens (n=71). All atheromas and obstructed old CABG
52
EFFECT OF CLARITHROMYCIN ON CHLAMYDIA PNEUMONIAE IN VASCULAR TISSUE
specimens showed histological signs of inflammation and advanced atherosclerosis
(thickened intima, plaques with thrombus, lymfocytes, foamcells, surface defects). The
other specimens showed normal to slightly thickened vascular walls.
The results of IHC are presented in table 2. C. pneumoniae MOMP antigen was
found in the majority of specimens of both study groups. Chlamydia LPS antigen was
found only once, in an atheroma from the placebo group. There was no significant
difference in the presence of antigens between the two groups. The real-time PCR and
the RUO-PCR failed to detect C. pneumoniae DNA in the 180 vascular specimens.
Table 2. Results of immunohistochemical staining of the vascular specimens obtained from the
study patientsa.
Clarithromycin
Placebo
p valueb
Positive specimens/
Positive specimens/
specimens tested, (%)
specimens tested, (%)
Coronary atheroma
MOMPc
10/14 (71.4)
10/17 (58.8)
0.47
LPSd
0/14 (0)
1/17 (5.9)
0.36
Old coronary grafte
MOMP
6/6 (100)
5/6 (83.3)
0.30
LPS
0/6 (0)
0/6 (0)
1.00
Mammary artery
MOMP
22/28 (78.6)
30/38 (78.9)
0.97
LPS
0/28 (0)
0/38 (0)
1.00
Saphenal vein
MOMP
21/32 (65.6)
32/39 (82.1)
0.11
LPS
0/32 (0)
0/39 (0)
1.00
Total
MOMP
59/80 (73.7)
77/100 (77.0)
0.69
LPS
0/80 (0)
1/100 (1.0)
1.00
Values are numbers (%);
a
more than one specimen was obtained from some patients;
b
a p-value of < 0·05 is considered statistically significant;
c
MOMP, C. pneumoniae major outer membrane protein antigen;
d
LPS, Chlamydia lipopolysaccharide antigen;
e
Obstructed old bypass graft from earlier CABG procedure.
Chlamydia IgG antibody titers were measured on inclusion (baseline serology) and 8
weeks after completion of treatment. Baseline serology was positive in 81.6% and 73.8%
of the patients in the clarithromycin group and the placebo group, respectively. The
corresponding percentages 8 weeks after treatment were 78.9% and 66.7%,
respectively. Clarithromycin had no significant effect on Chlamydia IgG antibody titers. In
both treatment groups, Chlamydia IgG antibody titers after treatment were not
significantly different to the baseline titers.
53
CHAPTER 4
DISCUSSION
Macrolide antibiotics, including clarithromycin, are active against C. pneumoniae. The
hypothesis that C. pneumoniae is a risk factor for atherosclerosis has lead to clinical
trials of macrolide treatment in patients with CAD8. Human placebo-controlled trials have
been performed to investigate the clinical effects of antibiotics in patients with vascular
disease, but they have not demonstrated whether antibiotics could achieve
microbiological cure in vascular tissue.
Our study was designed to examine whether clarithromycin treatment can
eradicate C. pneumoniae from vascular tissue of patients with CAD. We assessed the
effect of clarithromycin treatment on the presence of C. pneumoniae in vascular tissue
by IHC and two PCR assays. Also, we evaluated the effect of clarithromycin on
Chlamydia IgG titers.
In the present study, vascular specimens were tested by an automated real-time
PCR, which combines amplification, hybridization and quantitative product detection.
Moreover, the specimens were tested by an industry-developed RUO-PCR. However,
these two methods failed to detect C. pneumoniae DNA in any specimen. This indicates
that there is no evidence of acute C. pneumoniae infection in vascular tissue of CAD
patients. Other possible explanations for the negative results of the PCR assays could
be the low DNA concentration in the test samples and the patchy distribution of C.
pneumoniae DNA in vascular tissue14. An important difference between our study and
the study of Melissano et al.15. is the high detection rate of C. pneumoniae DNA in their
study. Melissano et al. evaluated the effect of roxithromycin on C. pneumoniae in carotid
atheromas. The authors concluded that roxithromycin treatment was effective in
eradicating C. pneumoniae from carotid atheromas since C. pneumoniae DNA was
detected in 31% (5/16) of the atheromas in the roxithromycin group and in 75% (12/16)
of the atheromas in the control group. However, they used one unstandardised
conventional semi-nested PCR assay to detect C. pneumoniae DNA. Conventional PCR
assays are unstandardised and known to produce conflicting results16,17. Also, taking the
limitations of the design of the study of Melissano et al. (open and not placebocontrolled) into account, the reliability of their results may be questioned. Efforts have
been recently focussed on the establishment of quantitative real-time PCR technology.
Although the real-time PCR and the RUO-PCR are not standardised, the first reports on
54
EFFECT OF CLARITHROMYCIN ON CHLAMYDIA PNEUMONIAE IN VASCULAR TISSUE
the evaluation of these tests suggest that they are sensitive, specific and
reproducible13,18.
Another finding of our study is the high prevalence of C. pneumoniae MOMP
antigen in vascular specimens. High detection rate of C. pneumoniae antigens has been
reported by others1. However, protocols of performance and interpretation of IHC are not
standardized 5. IHC requires a subjective reading and its interpretation remains difficult
because of background staining and nonspecific staining with antigenic components in
vascular specimens, such as inflammatory cells and tissue components19-21. It is also
possible that components of Chlamydia-like microorganisms described recently can
cause cross-reactivity to the monoclonal antibodies used in IHC staining9.
IHC detected C. pneumoniae MOMP antigen in 73.8% of specimens in the
clarithromycin group and in 77.0% of the specimens in the placebo group (p=ns).
Chlamydia LPS antigen was detected by IHC in only one specimen from the placebo
group. These results are consistent with the findings of other investigators. The
abundance of MOMP antigen and low detection rate of LPS antigen, in absence of C.
pneumoniae DNA in vascular specimens has been reported before9,22.
The conflicting results concerning the high detection rate of C. pneumoniae
MOMP antigen and the low detection rate of C. pneumoniae DNA might be partly
explained by the biology of Chlamydiae23. Alternation of activity and latency
characterizes infections with Chlamydiae. In advanced Chlamydiae infections, the
pathogen is no longer present, whereas its antigens can persist for a long time. The
persisting antigens may cause a cascade of events leading to fibrosis and scaring23.
In the present study, we could not demonstrate any effect of clarithromycin on the
presence of C. pneumoniae MOMP antigen in vascular tissue. Similar finding has been
demonstrated in animal models. Antibiotic treatment with azithromycin was not found
associated with elimination of chlamydial antigen from vascular specimens of rabbits
infected with C. pneumoniae24. Also, in a mice model, azithromycin treatment did not
affect the presence of C. pneumoniae in the aorta, lung, or spleen25.
Clarithromycin treatment had no effect on Chlamydia IgG antibody titers, and we
found no significant difference between baseline titers and the titers measured 8 weeks
after treatment. Circulating Chlamydia antibody titers have been used as a marker of
response to antibiotic treatment6. Gupta et al.6 reported a significant effect of
azithromycin on Chlamydia IgG antibody titers. However, our study supports the results
55
CHAPTER 4
of many other clinical trials that have shown that antibiotic therapy does not influence
Chlamydia antibody titers in patients with vascular disease26-30.
This is the first placebo-controlled, double-blind, randomised clinical trial that
assessed the effect of antibiotic treatment on the presence of C. pneumoniae in vascular
specimens. A major finding in this study was, that in vascular tissues of patients with
advanced CAD, no viable C. pneumoniae were present, but antigen debris of C.
pneumoniae. In the light of these findings, it can be questioned if one can demonstrate
the effect of antibiotics in patients with advanced atherosclerosis, since viable C.
pneumoniae is no longer present in vascular tissue. This may explain the results of many
clinical trials that failed to demonstrate any beneficial effect of antibiotic treatment in
patients with vascular disease26-32.
In conclusion, with use of different detection methods, no evidence could be
found for the presence of viable C. pneumoniae in vascular tissue of CAD patients.
Clarithromycin treatment of CAD patients had neither an effect on the presence of C.
pneumoniae antigens in vascular tissue nor an effect on circulating Chlamydia IgG
antibody titers. Antibiotic treatment of CAD patients is not indicated and it may not be
beneficial, since C. pneumoniae infection in these patients is probably not acute and
viable pathogens are no longer present in their vascular tissue. Future studies should be
focused on the effects of antibiotic treatment in patients within early stages of
atherosclerosis.
Acknowledgement
We thank Dr. Jerry Henslee (Abbott Laboratories) for making the LCx RUO-PCR
available for testing our materials. We thank Caroline de Jong, Ingrid Aarts and Marjolijn
Kerver for their technical assistance with the real-time PCR, and Jack Phillips (Abbott
Laboratories) for his technical assistance with the LCx RUO-PCR assay.
56
EFFECT OF CLARITHROMYCIN ON CHLAMYDIA PNEUMONIAE IN VASCULAR TISSUE
REFERENCES
1. Boman J, Hammerschlag MR. Chlamydia pneumoniae and atherosclerosis: critical
assessment of diagnostic methods and relevance to treatment studies.
Clin Microbiol Rev 2002;15:1-20
2. Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae as an emerging risk factor in
cardiovascular disease. JAMA 2002;288:2724-2731
3. Libua P, Karnani P, Personen E, et al. Endothelial dysfunction after repeated Chlamydia
pneumoniae infection in apolipoprotein E-knockout mice. Circulation 2000;102:1039-1044
4. Bloemenkamp DG, Mali WP, Visseren FL, van der Graaf Y. Meta-analysis of seroepidemiologic studies of the relation between Chlamydia pneumoniae and atherosclerosis:
does study design influence results? Am Heart J 2003; 145: 409-417
5. Dowell SF, Peeling RW, Boman J, et al. Standardizing Chlamydia pneumoniae assays:
recommendations from the Centers for Disease Control and Prevention (USA) and the
Laboratory Centre for Disease Control (Canada). Clin Infect Dis 2001;33:492-503
6. Gupta S, Leatham EW, Carrington D, Mendall MA, Kaski JC, Camm AJ. Elevated Chlamydia
pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of
myocardial infarction. Circulation 1997;96:404-407
7. Gurfinkel E, Bozovich G, Daroca A, Beck E, Mautner B. Randomised trial of roxithromycin in
non-Q-wave coronary syndromes: ROXIS Pilot Study. ROXIS Study Group. Lancet
1997;350:404-407
8. Higgins JP. Chlamydia pneumoniae and coronary artery disease: the antibiotic trials. Mayo
Clin Proc 2003;78:321-332
9. Meijer A, van der Vliet JA, Roholl PJM, Gielis-Proper SK, de Vries A, Ossewaarde JM.
Chlamydia pneumoniae in abdominal aortic aneurysms. Abundance of membrane
components in the absence of heat shock protein 60 and DNA. Arterioscler Thromb Vasc Biol
1999;19:2680-2686
10. Puolakkainen M, Parker J, Kuo CC, Grayston JT, Campbell LA. Further characterization of
Chlamydia pneumoniae specific monoclonal antibodies. Microbiol Immunol 1995;39:551-554
11. Tondella ML, Talkington DF, Holloway BP, et al. Development and evaluation of real-time
PCR-based fluorescence assays for detection of Chlamydia pneumoniae. J Clin Microbiol
2002;40:575-583
12. Harder TC, Harder M, et al. Characterization of phocid herpesvirus-1 and -2 as putative
alpha- and gammaherpesviruses of North American and European pinnipeds. J Gen Virol
1996;77:27-35
13. Chernesky M, Smieja M, Schachter J, et al. Comparison of an industry-derived LCx
Chlamydia pneumoniae PCR research kit to in-house assays performed in five laboratories. J
Clin Microbiol 2002;40:2357-2362
14. Cochrane M, Pospischil A, Walker P, Gibbs H, Timms P. Distribution of Chlamydia
pneumoniae DNA in atherosclerotic carotid arteries: significance for sampling procedures. J
Clin Microbiol 2003;41:1454-57
57
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15. Melissano G, Blasi F, Esposito G, et al. Chlamydia pneumoniae eradication from carotid
plaques. Results of an open, randomised treatment study. Eur J Vasc Endovasc Surg
1999;18:355-359
16. Apfalter P, Blasi F, Boman J, et al. Multicenter comparison trial of DNA extraction methods
and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens. J
Clin Microbiol 2001;39:519-524
17. Apfalter P, Assadian O, Blasi F, et al. Reliability of nested PCR for detection of Chlamydia
pneumoniae DNA in atheromas: results from a multicenter study applying standardized
protocols. J Clin Microbiol 2002;40:4428-4434
18. Apfalter P, Barousch W, Nehr M, et al. Comparison of a new quantitative ompA-based realTime PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory
specimens with four conventional PCR assays. J Clin Microbiol 2003;41:592-600
19. Ong G, Thomas BJ, Mansfield AO, Davidson BR, Taylor-Robinson D. Detection and
widespread distribution of Chlamydia pneumoniae in the vascular system and its possible
implications. J Clin Pathol 1996;49:102-106
20. Shor A, Philips JI, Ong G, Thomas BJ, Taylor-Robinson D. Chlamydia pneumoniae in
atheroma: consideration of criteria for causality. J Clin Pathol 1998;51:812-817
21. Taylor-Robinson D, Thomas BJ. Chlamydia pneumoniae in arteries: the facts, their
interpretation, and future studies. J Clin Pathol 1998;51:793-797
22. Meijer A, Roholl P J M, Gielis-Proper S K, Ossewaarde J M. Chlamydia pneumoniae
antigens, rather than viable bacteria, persist in atherosclerotic lesions. J Clin Pathol 2000;
53:911-916
23. Hoymans VY, Bosmans JM, Ieven M, Vrints CJ. Chlamydia pneumoniae and atherosclerosis.
Acta Chir Belg 2002;102:317-322
24. Muhlestein JB, Anderson JL, Hammond EH, et al. Infection with Chlamydia pneumoniae
accelerates the development of atherosclerosis and treatment with azithromycin prevents it in
a rabbit model. Circulation 1998;97:633-636
25. Rothstein NM, Quinn TC, Madico G, Gaydos CA, Lowenstein CJ. Effect of azithromycin on
murine arteriosclerosis exacerbated by Chlamydia pneumoniae. J Infect Dis 2001; 183:232238
26. Sinisalo J, Mattila K, Nieminnen MS, et al. The effect of prolonged doxycycline therapy on
Chlamydia pneumoniae serological markers, coronary heart disease risk factors and forearm
basal nitric oxide production. J Antimicrob Chemother 1998;41:85-92
27. Jackson LA, Stewart DK, Wang SP, Cooke DB, Cantrell T, Grayston JT. Safety and effect on
anti-Chlamydia pneumoniae antibody titres of a 1 month course of daily azithromycin in adults
with coronary artery disease. J Antimicrob Chemother 1999;44:411–414
28. Anderson JL, Muhlestein JB, Carlquist J, et al. Randomized secondary prevention trial of
azithromycin in patients with coronary artery disease and serological evidence for Chlamydia
pneumoniae infection: The Azithromycin in Coronary Artery Disease: Elimination of
Myocardial Infection with Chlamydia (ACADEMIC) study. Circulation 1999; 99:1540-1547
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29. Leowattana W, Bhuripanyo K, Singhaviranon L, et al. Roxithromycin inprevention of acute
coronary syndrome associated with Chlamydia pneumoniae infection: a randomized placebo
controlled trial. J Med Assoc Thai 2001;84 (suppl 3):S669-S675
30. O’Connor CM, Dunne MW, Pfeffer MA, et al. Azithromycin for the secondary prevention of
coronary heart disease events. The WIZARD study: a randomized controlled trial. JAMA
2003;290:1459-1466
31. Gurfinkel E, Bozovich G, Beck E, Testa E, Livellara B, Mautner B. Treatment with the
antibiotic roxithromycin in patients with acute non-Q-wave coronary syndromes. The final
report of the ROXIS Study. Eur Heart J 1999;20:121-127
32. Williams ES, Miller JM. Results from late-breaking clinical trial sessions at the American
College of Cardiology 51st Annual Scientific Session. J Am Coll Cardiol 2002;40:1-18
59
CHAPTER
5
EFFECT OF CLARITHROMYCIN ON INFLAMMATORY MARKERS IN
PATIENTS WITH ATHEROSCLEROSIS
HF Berg, B Maraha, GJ Scheffer, MF Peeters, JAJW Kluytmans
Clinical and Diagnostic Laboratory Immunology 2003; 10(4): 525-528
CHAPTER 5
SUMMARY
Atherosclerosis can to a certain extent be regarded as an inflammatory disease. Also,
inflammatory markers may provide information about the cardiovascular risk. Whether
macrolide antibiotics, especially clarithromycin, have an anti-inflammatory effect in
patients with atherosclerosis is not exactly known.
To study this phenomenon a placebo controlled, randomized, double blind study was
performed. 231 patients with documented coronary artery disease received a daily dose
of clarithromycin 500 mg slow release or placebo until the day of surgery. Inflammatory
markers (C-reactive protein [CRP], interleukin [IL]-2R, IL-6, IL-8, and tumour necrosis
factor (TNF)-α) were assessed during the pre-operative outpatient visit, on the day of
surgery, and 8 weeks after surgery. Also changes in the levels of inflammatory markers
between visits were calculated as delta.
Baseline patient characteristics were balanced between the two treatment groups:
average age was 66 years (SD=9.0), 79% of the patients were male, the average
number of used tablets was 16 (SD=9.3). The inflammatory markers of the groups as
well as the delta's were not significantly changed.
Treatment with clarithromycin did not influence the inflammatory markers in patients with
atherosclerosis.
62
EFFECT OF CLARITHROMYCIN ON INFLAMMATORY MARKERS
INTRODUCTION
Despite the use of pharmaceutical therapy against known risk factors and changes in
lifestyle and behaviour, cardiovascular disease remains a leading cause of death
worldwide1. Apart from well-known risk factors such as elevated and modified lowdensity protein (LDL) cholesterol, free radicals (caused by smoking), hypertension,
diabetes mellitus, genetic alterations, and hyperhomocysteinaemia, infections caused by
various
microorganisms
are
nowadays
also
considered
potential
causes
of
atherosclerosis. For example cytomegalovirus, Helicobacter pylori and Chlamydia
pneumoniae have been linked to the pathogenesis of atherosclerosis2. In addition,
atherosclerosis can be regarded as an inflammatory disease3.
All these risk factors can cause endothelial injury and dysfunction, which in many
studies is considered the first step in the pathogenesis of atherosclerosis. It forms the
basis of the so-called response-to-injury hypothesis4. This response to endothelial injury
is mediated at every stage of atherosclerosis by monocyte-derived macrophages and
specific subtypes of T lymphocytes3,4.
Elevated levels of C-reactive protein (CRP), tumour necrosis factor (TNF)-α or
interleukine (IL)-6 are all associated with an increased risk of
future myocardial
infarction5-8. Patients with an elevated CRP level (> 2.0 mg/L) are at risk of recurrent
angina pectoris and acute myocardial infarction (AMI). Conversely, in patients with
unstable angina pectoris the CRP level is elevated9. Researchers have now suggested
that inflammatory markers such as CRP may provide information about the
cardiovascular risk6.
Inflammatory markers such as IL-2R, IL-6, IL-8, TNF-α, and CRP can provide
information about the mechanism of the inflammatory reaction associated with
atherosclerosis. IL-2R, IL-6, IL-8, and TNF-α derive from activated monocytes,
macrophages, T-cells or endothelial cells. These inflammatory markers stimulate
fibroblasts and smooth muscle cells to proliferate. They may also induce free radical
generation by neutrophils, and this may facilitate oxidation of low-density protein
cholesterol and attract monocytes and other inflammatory cells to the area of endothelial
damage3,10. Cytokines promote the production of endogenous tissue plasminogen
activator and plasminogen activator inhibitor 1, which stimulate thrombus formation11.
There is much speculation about the anti-inflammatory potential of macrolides,
independent of their well-established role in the chemotherapy of infectious diseases.
63
CHAPTER 5
Macrolides have potentially relevant in-vitro, ex-vivo and in-vivo immunomodulatory
effects12. To investigate the effect of clarithromycin on inflammatory markers in patients
prior to coronary artery surgery, we performed a prospective, double-blind, randomized,
placebo controlled study.
MATERIALS AND METHODS
Between July 1999 and July 2001, patients with documented coronary artery disease
were enrolled in the study. All these patients were scheduled for coronary artery bypass
graft surgery. Patients were evaluated for inclusion in the study during a visit to the
preoperative outpatient clinic at the department of thoracic surgery of the Amphia
Hospital. Exclusion criteria included the following: concomitant administration of
terfenadine (Triludan), rifabutin (Mycobutin), cisapride (Propulsid), or antipyrine;
antibiotic therapy with a macrolide, tetracycline, or quinolone within 3 months prior to
inclusion in the study or during the study period; the presence of renal failure (serum
creatinine concentrations above 150 µmol/liter), elevated liver function (alanine
aminotransferase levels of >55 U/liter, aspartate aminotransferase levels of >45 U/liter,
total bilirubin levels of >27 µmol/liter, alkaline phosphatase levels of >180 U/liter); and for
female patients capable of childbearing, not taking adequate birth control precautions.
After the patients gave their informed consent, they were randomized in a double-blind,
placebo-controlled study. From that point on, they began receiving the study medication
until the day of surgery. The study medication consisted of either a daily dose of 500 mg
of slow-release clarithromycin (clarithromycin SR) or a matching placebo tablet (both
from Abbott laboratories, Ltd., Queenborough, Kent, England). Clarithromycin SR is well
absorbed following oral administration and well distributed in body fluids and tissues,
where it achieves high and persistent concentrations (half-life in tissue, 5.3 h).
From each patient, 10 ml of blood (extracted with EDTA) was obtained at the
following stages: (i) during the initial preoperative outpatient visit, (ii) on the day of
surgery just before the operation, and (iii) 8 weeks after surgery. Blood samples were
stored at 4°C immediately after collection and centrifuged within 2 h. Plasma was then
stored at -70°C pending further testing. This plasma was analyzed with an IMMULITE
analyzer according to the manufacturer’s instructions (EURO/DPC Ltd., Glyn Rhonwy,
United Kingdom) for quantitative estimation of soluble IL-2R (units per liter), IL-6
64
EFFECT OF CLARITHROMYCIN ON INFLAMMATORY MARKERS
(picograms per liter), IL-8 (picograms per liter), TNF-α (picograms per liter), and CRP
(milligrams per liter).
The study was approved by the local medical ethics committee.
Statistical analysis. A sample size calculation was made to measure the effect on the
CRP value based on the results from a previous study13. In this study the placebo group
had a mean CRP value of 8.7 mg/liter and a standard deviation (SD) of 6.0. For a
hypothesized reduction of 30% (2.6 mg/liter) of the CRP with a power of 90% and a
significance level of 0.05, 113 subjects were needed per study group.
All baseline characteristics were analyzed by using a Χ2 test for the distribution of
categorical variables and Student’s t test to compare continuous variables. To assess
the differences in inflammatory marker values between the two treatment groups, a
comparison was made using Student’s t test or the Mann-Whitney U test, when
appropriate. Statistical significance was accepted when P was <0.05.
RESULTS
A total of 231 patients were enrolled in the study. After randomization, 117 patients
received 500 mg of clarithromycin SR and 114 received a placebo. Table 1 shows that
the baseline patient characteristics were well balanced between the two treatment
groups. The mean numbers of tablets used before surgery were 16 (SD = 9.5) for the
clarithromycin group and 17 (SD = 9.6) for the placebo group. The mean age was 66
years (SD = 8.9) in the clarithromycin group versus 64 years (SD = 9.3) in the placebo
group. Eighty percent of the clarithromycin group was male versus 78.1% in the placebo
group. No significant differences were found.
Eight patients who during the study period simultaneously used antibiotics other
than a macrolide, tetracycline, or quinolone were excluded from this treatment analysis
due to possible interference from additive anti-inflammatory effects. The baseline levels
of inflammatory markers for both treatment groups on the day of the preoperative visit
(visit 1), as well as the values after treatment on the day of surgery (visit 2) and 8 weeks
after surgery (visit 3), are shown in Table 2. As the data in the table indicate, there were
no significant differences in the levels of inflammatory markers between the two groups
at visit 1, 2, or 3 (Table 2). Changes in the levels of inflammatory markers between visits
65
CHAPTER 5
were also calculated. There were no significant differences in these changes between
the treatment groups.
Table 1. Patient characteristics.
Value for group
Characteristica
Clarithromycin
(n=113)
Placebo
(n=110)
p-value
Age, yr
Weight, kg
Height, cm
NYHA
Pre-operative period (days)
Number of tablets used
66 (9.0)
82 (12.4)
172 (8.7)
2.8 (0.8)
17.7 (10.4)
16 (9.3)
64 (9.4)
80 (13.4)
172 (9.1)
2.8 (0.8)
20.4 (15.8)
17 (9.6)
NSb
NS
NS
NS
NS
NS
92 (81.4)
24 (21.2)
11 (9.7)
85 (77.3)
24 (21.8)
12 (10.9)
NS
NS
NS
8 (7.1)
13 (11.5)
66 (58.4)
8 (7.1)
6 (5.3)
10 (8.8)
108 (95.6)
54 (47.8)
4 (3.5)
54 (47.8)
21 (18.6)
8 (7.3)
8 (7.3)
70 (63.6)
3 (2.7)
5 (4.5)
7 (6.4)
110 (100)
49 (44.5)
3 (2.7)
56 (50.9)
18 (16.4)
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
86 (76.1)
20 (17.7)
83 (75.5)
17 (15.5)
NS
NS
66 (58.4)
113 (100)
0 (0)
69 (62.7)
109 (99.1)
3 (2.7)
NS
NS
NS
Medical history
Male
Smoker
COPD
Diabetes mellitis
IDDM
NIDDM
Dyslipidemia
Malignancy
Severe underlying disease
CVA
Angina pectoris
Myocardial infarction
Heart valve insufficiency
Hypertension
Earlier vascular surgery
Family history
Heart disease
Diabetes mellitus
Medication
Anti cholesterol
Anti hypertensives
Immunosuppressives
a NYHA, stage of heart failure according to the New York Heart Association classification; COPD, chronic
obstructive pulmonary disease; IDDM, insulin dependent diabetes mellitus; NIDDM, non-insulin dependent
diabetes mellitus; CVA, cerebrovascular accident. Values for medical and family histories and medication
are given as number (percent) of patients. Other values (for the first six characteristics) are given as means
(SDs).
b NS, not significant.
66
EFFECT OF CLARITHROMYCIN ON INFLAMMATORY MARKERS
Table 2. inflammatory markers of visit 1, 2 and 3.
Marker and
visit no.
CRP
1
2
3
TNF-α
1
2
3
IL2R
1
2
3
IL-6
1
2
3
IL-8
1
2
3
Treatment group (n)
a
Marker conc b
p-value
Mean
Std.dev.
Clarithromycin (113)
Placebo (108)
Clarithromycin (113)
Placebo (107)
Clarithromycin (109)
Placebo (108)
4.3
3.1
2.8
3.2
9.7
5.8
5.9
2.9
3.2
7.2
20.7
8.2
0.29c
Clarithromycin (113)
Placebo (108)
Clarithromycin (113)
Placebo (107)
Clarithromycin (109)
Placebo (106)
90.8
130.1
263.5
313.5
157.1
154.5
237.3
272.9
327.6
327.6
305.6
308.3
0.25
Clarithromycin (113)
Placebo (109)
Clarithromycin (112)
Placebo (107)
Clarithromycin (108)
Placebo (107)
547.6
476.9
418.6
351.7
601.0
533.2
717.6
200.8
683.0
220.8
672.6
236.9
0.20c
Clarithromycin (112)
Placebo (109)
Clarithromycin (112)
Placebo (104)
Clarithromycin (107)
Placebo (106)
30.7
33.1
24.7
46.3
15.6
40.2
149.3
158.9
133.5
193.8
96.1
176.2
0.91
Clarithromycin (113)
Placebo (109)
Clarithromycin (113)
Placebo (107)
Clarithromycin (108)
Placebo (107)
25.4
6.8
20.5
7.3
14.2
8.8
116.8
12.8
92.2
16.6
66.3
21.5
0.67c
0.61
0.15c
0.29
0.95
0.33
0.33
0.34
0.93c
0.47c
0.42
a n, number of study patients out of a total of 223.
b Concentrations are given in milligrams per liter (CRP), picograms per liter
(TNF-α, IL-6, and IL-8), and units per liter (IL-2R).
c Determined by the Mann-Whitney test.
DISCUSSION
Treatment with a daily dose of 500 mg of clarithromycin SR in patients with coronary
artery disease prior to cardiac surgery did not significantly alter the inflammatory markers
67
CHAPTER 5
directly after treatment on the day of surgery or 8 weeks after surgery. Clarithromycin
can therefore be considered to have no or little anti-inflammatory potential.
The hypothesis that atherosclerosis has an infectious etiology has promoted
studies of the effect of antibiotics, especially macrolides. The activity against C.
pneumoniae and the general anti-inflammatory effect on the outcome in patients with
cardiovascular disease were studied retrospectively as well as prospectively. For
example, Østergaard et al. in a retrospective cohort study14 assessed the timedependent effect of macrolide therapy versus that of penicillin therapy on the risk of
hospitalization due to cardiovascular disease. The authors concluded that the decreased
relative risk of hospitalization (0.48) due to cardiovascular disease in users of macrolides
within 3 months indicated a possible direct anti-inflammatory effect on diseased vessels,
but this was not further studied. In several prospective intervention studies using either
azithromycin (AZ) or roxithromycin, the anti-inflammatory effect was examined. Gupta et
al.15,16 performed the first intervention trial in which 60 survivors of acute myocardial
infarction with persistently elevated antichlamydial antibody titers were treated with a 3day course of AZ, followed by a single dose per week for 3 months, or with a placebo. In
a subgroup of patients treated with a double dose of AZ, significant decreases in total
monocyte tissue factor and CD11b were found after 6 months (but not 3 months). This
indicates a stronger anti-inflammatory effect after 6 months when a double dose of AZ
was used. However, no significant differences were noted in the levels of any of the
inflammatory markers in either of the normal groups receiving AZ or placebo. In a similar
study by Gurfinkel et al.17, 202 patients with coronary heart disease were treated with
roxithromycin for 30 days. CRP levels decreased in both the placebo and the
roxithromycin treatment groups at 30 days, but the declines did not reach statistical
significance. Moreover, CRP was not associated with the significant reduction in
ischemic events. This result indicates that the reduction in cardiovascular events in these
studies is based on a stabilizing effect on atherosclerotic plaques, caused by an
antichlamydial effect, rather than on an anti-inflammatory effect.
More recently, Anderson et al.13 treated 302 patients who had coronary artery
disease and a seropositive reaction to C. pneumoniae with AZ or placebo (in a similar
treatment protocol as that described by Gupta et al.15,16). The levels of four inflammatory
markers were unchanged at 3 months but showed minor changes after 6 months. The
changes in the levels of inflammatory markers before and after treatment were
significantly lower for CRP (p = 0.011) and IL-6 (p = 0.043) after 6 months (not after 3
68
EFFECT OF CLARITHROMYCIN ON INFLAMMATORY MARKERS
months). It must be stressed that no difference was found in clinical events and that
these results are based on minor changes; a major anti-inflammatory effect was not
shown. The overall minor changes in inflammatory markers are consistent with our study
results.
The duration of treatment could be of critical importance in explaining the
different results in various studies. Our study patients were treated, on average, with 16
tablets (a number equivalent to the number of treatment days). We therefore repeated
our analysis on a subgroup of 114 patients who were treated with 14 or more tablets, but
we found no differences in levels of inflammatory markers between the two groups at
visit 1, 2 or 3. A repeated analysis of a small subgroup of 56 patients treated with 21 or
more tablets also did not show any differences. For that reason we can conclude that the
possible anti-inflammatory effect of clarithromycin, if any at all, does not increase with
the duration of treatment. In addition, in the intervention studies mentioned above, even
though patients received long-term treatment for 1 to 3 months13,15,16, such treatment had
little or no effect on inflammatory markers. Other data about a difference between the
effects of short-term and long-term administration of macrolides on the immune
response have been reported in a review by Labro12. These studies are, however, mainly
ex vivo studies, so comparison to the results with our study population is not appropriate.
In conclusion, several studies have demonstrated an anti-inflammatory effect of
macrolides in vitro and in treatment of diseases involved with the bronchial
epithelium18,19,20. The present study is, however, in our opinion the first reported in vivo
study about the effect of clarithromycin in patients with atherosclerosis and indicates that
clarithromycin has no measurable anti-inflammatory effect in such patients.
69
CHAPTER 5
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2. Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link?
Lancet 1997;350:430-436
3. Hansson GK, Libby P, Schönbeck U, Yan ZQ. Innate and adaptive immunity in the
pathogenesis of atherosclerosis. Circulation Research 2002;91:281-291
4. Ross R, Glomset JA. Atherosclerosis and the arterial smooth muscle cell: proliferation of
smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science
1973;180:1332-1339
5. Lindmark E, Diderholm E, Wallentin L, Siegbahn A. Relationship between interleukin 6 and
mortality in patients with unstable coronary artery disease. JAMA 2001;286:2107-2113
6. Ridker PM. High-sensitivity C-reactive protein. Potential adjunct for global risk assessment in
the primary prevention of cardiovascular disease. Circulation 2001;103:1813-1818
7. Ridker PM, Rifai N, Pfeffer M, Sacks F, Lepage S, Braunwald E, for the cholesterol and
recurrent events (CARE) investigators. Elevation of tumour necrosis factor-α and increased
risk of recurrent coronary events after myocardial infarction. Circulation 2000;101:2149-2153
8. Ridker PM, Rifai N, Stampfer MJ, Hennekens CH. Plasma concentration of interleukin-6 and
the risk of future myocardial infarction among apparently healthy men. Circulation
2000;101:1767-1772
9. Liuzzo G, Biasucci LM, Gallimore JR, Grillo RL, Rebuzzi AG, Pepys MB, Maseri A. The
prognostic value of C-reactive protein and serum amyloid A protein in severe unstable
angina. N Eng J Med 1994;331:417-424
10. Gupta S, Gamm AJ. Chlamydia pneumoniae and coronary heart disease. BMJ
1997;314:1778-1779
11. Mehta JL, Saldeen TGP, Rand K. Interactive role of infection, inflammation and traditional risk
factors in atherosclerosis and coronary artery disease. J Am Coll Cardiol 1998;31:1217-1225
12. Labro MT. Anti-inflammatory activity of macrolides: a new therapeutic potential? J Antimicrob
Chemother 1998;41:Suppl.B:37-46
13. Anderson JL, Muhlestein JB, Carlquist J, Allen A, Trehan S, Nielson C, Hall S, Brady J, Egger
M, Horne B, Lim T. Randomized secondary prevention trial of azithromycin in patients with
coronary artery disease and serological evidence for Chlamydia pneumoniae infection: the
azithromycin in coronary artery disease: Elimination of myocardial infection with Chlamydia
(ACADEMIC) study. Circulation1999;99:1540-1547
14. Østergaard L, Sørensen HT, Lindholt J, Sørensen TE, Pedersen L, Eriksen T, Andersen PL.
Risk of hospitalization for cardiovascular disease after use of macrolides and penicillins: a
comparative prospective cohort study. J Infect Dis 2001;183:1625-1630
15. Gupta S, Leatham EW, Carrington D, Mendall M, Ireson N, Tooze J, Bevan D, Camm AJ,
Kaski JC. The effect of azithromycin in post-myocardial infarction (MI) patients with elevated
Chlamydia pneumoniae antibody titres. J Am Coll Cardiol 1997;29:209A. Abstract
70
EFFECT OF CLARITHROMYCIN ON INFLAMMATORY MARKERS
16. Gupta S, Leatham EW, Carrington D, Mendall MA, Kaski JC, Camm AJ. Elevated Chlamydia
pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of
myocardial infarction. Circulation 1997;96:404-407
17. Gurfinkel E, Bozovich G, Beck E, Testa E, Livellara B, Mautner B. Treatment with the
antibiotic roxithromycin in patients with acute non-Q-wave coronary syndromes. The final
report of the ROXIS Study. Eur Heart J 1999;20(2):121-127
18. Amayasu H, Yoshida S, Ebana S, Yamamoto Y, Nishikawa T, Shoji T, Nakagawa H,
Hasegawa H, Nakabayashi M, Ishizaki Y. Clarithromycin suppresses bronchial
hyperresponsiveness associated with eosinophilic inflammation in patients with asthma. Ann
Allergy Clin Immunol 2000;84:594-598
19. Avila PC, Boushey HA. Macrolides, asthma, inflammation, and infection. Ann Allergy Clin
Immunol 2000;84:565-568
20. Sakito O, Kadota J, Kohno S, Abe K, Shirai R, Hara K. Interleukin 1 beta, tumor necrosis
factor alpha, and interleukin 8 in bronchoalveolar fluid of patients with diffuse panbronchiolitis:
a potential mechanism of macrolide therapy. Respiration 1996;63:42-48
71
CHAPTER
6
TREATMENT WITH CLARITHROMYCIN PRIOR TO CORONARY ARTERY
BYPASS GRAFT SURGERY DOES NOT PREVENT SUBSEQUENT CARDIAC
EVENTS
HF Berg, B Maraha, GJ Scheffer, M Quarles-van Ufford, CMJE Vandenbroucke-Grauls,
MF Peeters, JAJW Kluytmans
Submitted
CHAPTER 6
SUMMARY
Recently, Chlamydia pneumoniae has been identified to play a possible role in the
pathogenesis of atherosclerosis. We investigated whether treatment with clarithromycin
prior to coronary artery bypass graft surgery (CABG) would prevent subsequent
cardiovascular events and mortality.
473 patients, who were scheduled for CABG, were randomly assigned to receive either
clarithromycin (CL) or placebo (PB) until the day of surgery in a double-blind trial.
Duration of treatment was on average 16 days. During the 2 years follow-up, mortality
and cardiovascular events were assessed. Follow-up at 2 years could be achieved in
89.6 % of all patients. Baseline patient characteristics were well balanced between the
two treatment groups. Mortality was equal in both groups: 5 (2.4%) in the CL-group and
6 (2.8%) in the PB-group (relative risk (RR), 0.87; 95% CI, 0.27 to 2.79; p=0.81). Also,
there were no significant differences in cardiovascular event rates during the follow-up
period (RR 0.91; 95%CI, 0.50 to 1.64; p=0.86). The overall event rate was 10.6%.
Treatment with clarithromycin in patients scheduled for CABG did not reduce the
subsequent occurrence of cardiovascular events or mortality during a 2-year follow-up.
74
TREATMENT WITH CLARITHROMYCIN: A TWO YEAR FOLLOW-UP
INTRODUCTION
It is generally acknowledged that atherosclerosis can be considered partly an
inflammatory disease. It may result from an immune response to various inflammatory
stimuli, leading to atherogenesis and coronary artery disease (CAD) progression1.
Common chronic infections, caused by viruses and bacteria, may contribute to this
inflammatory process and interact with classical cardiovascular risk factors such as
smoking, hypercholesterolaemia, diabetes mellitus, hypertension and a positive family
history of CAD1,2. To date Chlamydia (C.) pneumoniae is the leading potential 'culprit'
infectious agent with the strongest evidence linking infection and atherosclerosis3.
Seroepidemiological studies, detection of the organism within atherosclerotic vascular
tissue (culture, electron microscopy, antigen and DNA detection techniques), in vitro
experiments and animal models have supported a potential role for this micro-organism
in the pathogenesis of atherosclerosis4,5. Recently, studies on the reliability of molecular
detection methods have raised serious questions regarding the interpretation of previous
studies6. Therefore, the exact role of C. pneumoniae remains to be determined. The
chain of evidence could be strengthened by studying the effect of antibiotic treatment on
clinical outcome in patients with CAD.
World-wide interest was generated by the positive results of two smaller studies
published in 19977,8, which showed a better outcome after treatment with a macrolide.
The purpose of the present study was to determine the effect of treatment with the
macrolide antibiotic clarithromycin prior to coronary artery bypass graft surgery (CABG)
on clinical outcome and prognosis in patients with severe atherosclerosis in a doubleblind, randomised, placebo-controlled study.
METHODS
Patients.
Between July 1999 and July 2001, patients with documented coronary
artery disease were enrolled in the study. All patients were scheduled for coronary artery
bypass graft (CABG) surgery. Inclusion was carried out during attendance of the preoperative outpatient clinic at the department of thoracic surgery of the Amphia Hospital.
Exclusion criteria were: participation in another study, concomitant administration
of terfenadine, rifabutin, cisapride or antipyrine; antibiotic therapy with a macrolide,
75
CHAPTER 6
tetracycline, or quinolone within three months prior to inclusion or during the study
period; the presence of renal failure (serum creatinine rates above 150 µmol/l), elevated
liver function (ALAT > 55 U/l, ASAT > 45 U/l, total bilirubin > 27 µmol/l, alkaline
phosphatase
> 180 U/l), female patients capable of child-bearing but not taking
adequate birth control precautions.
The study was approved by the local Medical Ethics Committee.
The research physician saw all patients during their visit to the preoperative
outpatient clinic, examined their patient files, interviewed them and asked for informed
consent. Patients were randomised in a double-blind, placebo controlled study. The
study medication consisted of either a daily dose of clarithromycin 500 mg slow release
(CL) or a placebo tablet (PB). Clarithromycin SR and matching placebo tablets were
obtained from Abbott laboratories, (Abbott Laboratories Ltd, Queenborough, Kent,
England ME11 5EL). Treatment duration was from randomisation until day of surgery.
An independent pharmacist dispensed either CL or PB tablets according to a
computer generated randomisation table, which stratified patients in groups of 10. The
research physician responsible for seeing the patients allocated the next available
number on entry into the trial, and each patient received the corresponding tablets
directly from the researcher. The code was revealed to the researcher after the 2.5 years
of follow-up was completed
Outcome data.
Data on clinical outcome and cardiovascular events were collected
by the research physician by telephone interview of each patient at 6 and 12 months
after cardiac surgery, and by interviewing the general practitioner at the end of the 2
years of follow-up. The primary end point was overall mortality. Secondary end points
were: reappearance of angina pectoris, myocardial re-infarction, percutaneous coronary
re-intervention or coronary artery bypass surgery, stroke, and peripheral artery disease
that required bypass or percutaneous intervention.
Statistical analysis.
All base-line characteristics were analysed with a χ2-test or a Student’s t test
when appropriate. Statistical analysis was performed using SPSS, version 11.0.
Statistical significance was accepted when P<0.05.
76
TREATMENT WITH CLARITHROMYCIN: A TWO YEAR FOLLOW-UP
641 total eligible patients
168 excluded
30 other surgical procedure
22 did not understand the study protocol
52 refused participation
29 use of macrolides, quinolones, or
tetracyclines ≤ 3 months ago
22 participated in other trials
2 liver disease (elevated liver enzymes)
7 renal disease (elevated kreatinin)
473 randomly allocated
238 clarithromycin
235 placebo
16 elective termination
2 died before surgery
1 pre-operative stroke
7 elective termination
2 died before surgery
2 pre-operative stroke
219 completed treatment
224 completed treatment
11 concomitant use of other
antibiotics
4 concomitant use of other
antibiotics
4 other therapy than CABG
208 analysed for primary
outcome
216 analysed for primary
outcome
Figure 1. Trial profile.
RESULTS
Between July 1999 and July 2001, 641 patients were seen at the pre-operative
outpatients’ clinic. The trial profile (figure 1) shows that 473 patients were enrolled in the
77
CHAPTER 6
study. Complete follow-up data were obtained on 424 patients. Finally, 208 patients of
the CL group and 216 patients of the PB group were analysed.
Table 1 shows that the patient characteristics were well balanced between the
two treatment groups. No significant differences were found. Patients were treated on
average for 16 days. There were no significant differences in the event rates during the
follow-up period, which is shown in table 2. Total mortality was almost equal for both
treatment groups: 5 (2.4%) in the CL group and 6 (2.8%) in the PB group (relative risk,
0.87; 95% CI, 0.27 to 2.79; p=0.81). If only patients were analysed who had been treated
for 21 days or more (138 patients), event rates were also not different between the
treatment groups (total mortality 3 (5.1%) in CL and 1 (1.5%) in PB group).
Table 1. Baseline patient characteristics and medical history.
Clarithromycin
Placebo
(n=208)
(n=216)
Baseline characteristics
Mean
SD
Mean
SD
Age
Weight
Length
NYHA-scorea
Number of treatment days
64.6
82.3
172.1
2.85
16.0
Number
166
43
8.8
13.5
11.5
0.8
7.9
%
78.8
20.7
64.0
81.1
171.0
2.88
16.6
Number
169
42
10.9
14.3
11.0
0.8
7.7
%
78.2
19.4
119
21
12
18
123
10
17
200
107
7
81
47
57.2
10.1
5.8
8.7
59.1
4.8
8.2
96.2
51.4
3.4
38.9
22.6
130
22
13
25
136
6
16
214
104
3
90
47
60.2
10.2
6.0
11.6
63.0
2.8
7.4
99.1
48.1
1.4
41.7
21.8
161
31
77.4
14.9
157
32
72.7
14.8
128
208
61.5
100
143
215
66.2
99.5
Sex (male)
Smoker (currently)
Medical history
Smoker in past
COPDb
Diabetes Mellitus Type I
Type II
Hypercholesterolaemia
Malignancy
Stroke
Angina pectoris
Myocardial infarction
Heart valve insufficiency
Hypertension
Earlier vascular surgery
Family medical history
Cardiovascular disease
Diabetes mellitus
Medication
Statins
Anti-hypertensive drugs
All baseline characteristics were not significantly different between the treatment groups
a
angina pectoris score by classification of the New York Heart Association
b
Chronic Obstructive Pulmonary Disease
78
TREATMENT WITH CLARITHROMYCIN: A TWO YEAR FOLLOW-UP
Table 2. Clinical Events during 24-month follow-up.
Events
Clarithromycin
(n=208)
Placebo
(n=216)
P
Relative Risk
(95% CI)
Death
5 (2.4%)
6 (2.8%)
1.0
0.87 (0.23 - 3.15)
(Unstable) angina pectoris
Nonfatal MI
Revascularizationa
7 (3.4%)
1 (0.5%)
8 (3.8%)
11 (5.1%)
3 (1.4%)
4 (1.9%)
0.52
0.64
0.35
0.66 (0.24 - 1.80)
0.35 (0.01 - 3.67)
2.08 (0.58 - 8.11)
Overall cardiac events
16 (7.6%)
18 (8.3%)
0.95
0.92 (0.46 - 1.85)
Stroke
Peripheral vascular surgery
8 (3.8%)
1 (0.5%)
3 (1.4%)
2 (0.9%)
0.20
1.0
2.77 (0.68 - 13.05)
0.52 (0.02 - 7.21)
Sternal wound infection
1 (0.5%)
1 (0.5%)
1.0
1.04 (0.03 - 37.76)
a
Percutaneous coronary intervention or coronary bypass surgery
DISCUSSION
Treatment of patients with CAD with a daily dose of 500 mg clarithromycin SR for 16
days on average prior to cardiac surgery (CABG) did not reduce rates of mortality nor
cardiovascular events during two years after surgery. A course of clarithromycin in
patients with severe atherosclerosis appears therefore to be of no clinical value.
Randomized controlled trials have been performed to analyze the effect of
treating patients with CAD and its associated complications with antibiotics that have
activity against C. pneumoniae. These trials were undertaken after several investigators
found an association of C. pneumoniae with CAD. This association was first noted in
1988 by Saikku et al in Finland9. Further interest in the association was mainly based on
the results of seroepidemiological studies, although not all studies or cohorts have found
a significant relationship after correcting for confounders10. More evidence was gathered
when the presence of C. pneumoniae in some atherosclerotic lesions was shown. Also
animal model experiments suggested C. pneumoniae-induced atherogenesis4,5,11.
Since the promising results of two smaller clinical treatment studies in 19977,8,
several larger intervention trials have been performed (table 3). Thus far, the significant
effects in some small clinical trials
trials
14-20,29
7,8,12,13
have not been confirmed by large randomized
, including the results found in our study. Antibiotic treatment regimes and
duration of follow-up varied significantly between the studies, as well as the size and
type of the study populations (table 3). Nevertheless, the majority of randomised79
CHAPTER 6
controlled trials did not show any beneficial effect in outcome after a course of
macrolides in patients with established coronary artery disease.
There are several possibilities why we did not detect a significant difference
between the two groups. The duration of treatment in our study might have been too
short and therefore not effective enough. In the CLARIFY study, Sinisalo et al.
administered clarithromycin for 3 months on patients with acute non-Q-wave infarction or
unstable angina and reduced the risk of subsequent cardiac events by 41%12. Other
trials do not indicate that the duration of treatment is an important item (table 3).
Especially the WIZARD study, with 7747 patients the largest study by far, treated for 77
days and did not find a significant effect19. When, in our study, a subgroup of patients
treated for 21 days was analysed, no difference was found. Therefore, it is unlikely that
the duration of treatment played an important role.
The power of the study could also be a point of discussion. However, there was
no trend towards a better or worse outcome. The most important variables, being
mortality and all cardiac events, were nearly equal in both groups. Also other large
studies like AZACS18 and WIZARD19, which included 1439 and 7747 patients
respectively, did not detect a reduction in cardiovascular events.
Duration of follow-up and type of antibiotic also seem of no significant influence
indicated by contrasting outcome of these clinical trials (table 3). It is remarkable that the
positive results of smaller clinical trials are not found in the larger ones. Off course this
could be explained by the fact that they are coincident findings. Smaller trials may have
studied a more selective population, which indicates that certain groups of individuals
might benefit from antibiotics. In the WIZARD trial19, analysis within subpopulations
showed trends toward a favorable effect of antibiotic therapy in men who smoke or who
have diabetes or hypercholesterolemia. In a post hoc analysis of the ISAR-3 trial21,
patients with the highest titres of C. pneumoniae antibodies had a reduced rate of
restenosis if given roxithromycin than if given placebo. In our study, treatment with
clarithromycin did not differ between patients who have diabetes or have not, and those
who smoke, or do not.
The negative results of the majority of recent trails, including ours, challenge the
hypothesis whether viable C. pneumoniae plays a causal role in the pathogenesis of
atherosclerosis. This doubt is confirmed by other signals in the research field. In more
recent studies many investigators have failed to detect C. pneumoniae DNA in vascular
specimens of patients with atherosclerosis22-26. Major inter-laboratory differences exist,
80
TREATMENT WITH CLARITHROMYCIN: A TWO YEAR FOLLOW-UP
especially concerning false-positivity. Moreover, there is no established reference
method, which makes it almost impossible to draw clear conclusions about the presence
of C. pneumoniae in vascular tissue and therefore its association with atherosclerosis6.
In addition, the results of a recent meta-analysis appear to indicate that there is no clear
relation between chlamydial IgG and atherogenesis10. Whether infection with C.
pneumoniae plays a putative role in the development of atherosclerosis needs therefore
to be questioned. Analysis of the pooled cardiac events of all studies shown in table 3
did not show a significant effect for macrolide treatment (RR 0.97; 95% CI: 0.89 – 1.06).
This was the same for the pooled mortality (RR 0.97; 95%CI: 0.82 – 1.16). The results of
our study are in line with these pooled data. Therefore, at present no recommendation
for the use of antibiotic therapy for the secondary prevention of atherosclerosis can be
made.
Apart from its ability to treat C. pneumoniae infection and to reduce the
vasotropic infectious burden, an anti-inflammatory effect of macrolide antibiotics is often
mentioned. However, an anti-inflammatory effect of clarithromycin as measured by the
levels of cytokines and acute phase proteins was not detected in our study patients, as
published previously27.
The use of macrolide antibiotics deserves serious thought. In our study
population, a treatment of 16 days with clarithromycin caused a marked effect on the
development of resistance in the oropharyngeal flora that lasted for at least 8 weeks
28
.
Therefore, macrolides should be used only when the indication is clearly established.
In conclusion, this study shows that treatment with macrolide antibiotics has no
beneficial effect on mortality, cardiac events and surgical site infection in patients with
severe atherosclerosis undergoing cardiac surgery. This provides further evidence
against a causative role of C. pneumoniae in the development of atherosclerosis. The
relationship between C. pneumoniae and atherosclerosis should be studied in more
detail before new therapeutic trails are to be performed.
81
82
Gurfinkel
1999
Leowattana
2001
Muhlestein
2000
20
15
29
14
Gurfinkel
1997
Anderson
1999
10
Gupta
1997
9
Author
b
c
c
Siriraj Hospital
trial, Bankok
ACADEMIC
ACADEMIC
ROXIS
ROXIS
b
St George’s
Hospital trial
Name
Azithromycin
Roxithromycin
302 patients with
coronary artery
disease with anti-C.
pneumoniae titer
84 patients with
acute coronary
syndrome
Roxithromycin
202 patients with
unstable angina or
non-Q-wave MI
Azithromycin
Roxithromycin
202 patients with
unstable angina or
non-Q-wave MI
302 patients with
coronary artery
disease with anti-C.
pneumoniae titer
Roxithromycin
Antibiotic
220 male survivors
of MI
Population
30
90
90
30
30
3-6
Duration
in days
3
24
6
6
1
18 +/- 4
Follow-up
in months
Fourfold decreased risk
cardiovascular events in
patients with anti-C.
pneumoniae titer ≥ 1/64
Statistical significant
reduction in the primary
composite triple endpoint
rates in the treatment group
Secondary triple combined
event rate was not
significantly different at 6
months
No significant difference in
the primary end point
between the 2 groups
No significant difference in
the primary end point
between the 2 groups
No significant difference in
cardiac events
Primary: cardiac ischemic
death, MI, severe recurrent
ischemia
Secondary: anti-C.
pneumoniae titer, CRP
Primary: cardiac ischemic
death, MI, severe recurrent
ischemia
Secondary: anti-C.
pneumoniae titer, CRP
Primary: cardiovascular
events (cardiac death, MI,
stroke, hospitalization for
unstable angina,
resuscitated cardiac arrest,
revascularization)
Secundary: inflammatory
markers
Primary: cardiovascular
events (cardiac death, MI,
stroke, hospitalization for
unstable angina,
resuscitated cardiac arrest,
revascularization)
Secundary: inflammatory
markers
Primary: cardiac death,
unplanned
revascularization, recurrent
angina, MI
Secondary: anti-C.
pneumoniae titer
Results
Primary: nonfatal MI,
unstable angina,
cardiovascular death
Secondary: anti-C.
pneumoniae titer
End point
Table 3. Randomised Controlled Intervention Trials in patients with coronary artery disease.
1.01
(0.56 - 1.84)
0.89
(0.51 - 1.57)
1.12
(0.45 – 2.76)
0.60
(0.24 - 1.44)
0.11
(0.01 – 0.82)
0.27
(0.06 - 0.95)
a
RR (95%CI)
83
13
12
19
Clarithromycin
473 patients with
angina or a history
of MI scheduled for
CABG
Amphia
Hospital,
Breda
This study
2004
Relative Risk of combined cardiovascular end points
MI indicates myocardial infarction
b
This study was published twice, after 1 and 6 months of follow-up
c
This study was published twice, after 6 and 24 months of follow-up
a
Azithromycin
AZACS
Cercek
2003
Azithromycin
7747 patients with
a history of MI
longer than 6
weeks previously
1439 patients with
unstable angina or
acute MI
18
ANTIBIO
Clarithromycin
148 patients with
acute non-Q-wave
infarction or
unstable angina
Roxithromycin
17
WIZARD
CLARIFY
STAMINA
Azithromycin/
Amoxicillin
325 patients with
acute MI or instable
angina (acute
coronary
syndromes)
872 patients with
an acute MI
Zahn
2003
O’Connor
2003
Sinisalo
2002
Stone
2002
16
5
42
77
90
7
24
6
12
48
18 +/- 12
12
No difference in events at 6
months follow-up
No difference in events at 24
months follow-up
Primary: overall mortality
Secondary: reappearance
angina, re-MI, PTCA/
CABG, stroke, peripheral
vascular surgery
0.92
(0.46-1.85)
1.03
(0.82-1.29)
1.21
(0.95-1.53)
No difference in clinical
events. Death, MI,
resuscitation, stroke, unstable
angina was not significantly
different at 12-months followup
Primary: death, recurrent
MI, revascularization
Secondary: unstable
angina, congestive heart
failure requiring admission
0.93
(0.83 - 1.05)
0.49
(0.26 - 0.92)
0.61
(0.41 - 0.93)
No significant difference in
end points. Baseline titer of
IgG antibodies against C.
pneumoniae had no effect on
outcome
Significant reduction
cardiovascular events
throughout mean follow-up
Primary: death, MI, angina
Secondary: death, MI,
unstable angina, ischemic
stroke, critical limb
ischemia
Primary: death from any
cause, nonfatal re-MI,
coronary revascularization,
hospitalization for angina
Secondary: anti-C.
pneumoniae titer, CRP,
TNF-α, fibrinogen
Primary: total mortality
Secondary (combined):
death, reinfarction,
resuscitation, stroke,
postinfarction angina until
hospital discharge,
unstable angina leading to
hospitalization, rate of
PTCA or CABG
intervention
36% reduction of all end
points in patients receiving
antibiotics
Primary: unstable angina,
MI
Secondary: antibodies to
C. pneumoniae and H.
pylori
CHAPTER 6
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13. Stone AFM, Mendall MA, Kaski JC, Edger TM, Risley P, Poloniecki J, Camm AJ,
Northfield.TC. Effect of Treatment for Chlamydia pneumoniae and Helicobacter pylori on
Markers of Inflammation and Cardiac Events in Patients With Acute Coronary Syndromes:
South Thames Trial of Antibiotics in Myocardial Infarction and Unstable Angina (STAMINA).
Circulation 2002; 106:1219-1223
14. Gurfinkel E, Bozovich G, Beck E, Testa E, Livellara B, Mautner B. Treatment with the
antibiotic roxithromycin in patients with acute non-Q-wave coronary syndromes. The final
report of the ROXIS Study. Eur Heart J 1999;20(2):121-127
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TREATMENT WITH CLARITHROMYCIN: A TWO YEAR FOLLOW-UP
15. Muhlestein JB, Anderson JL, Carlquist JF, Salunkhe K, Horne BD, Pearson RR, Bunch TJ,
Allen A, Trehan S, Nielson C. Randomized secondary prevention trial of azithromycin in
patients with coronary artery disease: primary clinical results of the ACADEMIC study.
Circulation 2000;102(15):1755-1760
16. Sander D, Winbeck K, Klingelhofer J, Etgen T, Conrad B. Reduced progression of early
carotid atherosclerosis after antibiotic treatment and Chlamydia pneumoniae seropositivity.
Circulation 2002;106(19):2428-2433
17. Zahn R, Schneider S, Frilling B, Seidl K, Tebbe U, Weber M, Gottwik M, Altmann E, Seidel F,
Rox J, Hoffler U, Neuhaus KL, Senges J; Working Group of Leading Hospital Cardiologists.
Antibiotic therapy after acute myocardial infarction: a prospective randomized study.
Circulation 2003;107(9):1253-1259
18. Cercek B, Shah PK, Noc M, Zahger D, Zeymer U, Matetzky S, Maurer G, Mahrer P; AZACS
Investigators. Effect of short-term treatment with azithromycin on recurrent ischaemic events
in patients with acute coronary syndrome in the Azithromycin in Acute Coronary Syndrome
(AZACS) trial: a randomised controlled trial. Lancet 2003;361:809-813
19. O'Connor CM, Dunne MW, Pfeffer MA, Muhlestein JB, Yao L, Gupta S, Benner RJ, Fisher
MR, Cook TD; Investigators in the WIZARD Study. Azithromycin for the secondary prevention
of coronary heart disease events: the WIZARD study: a randomized controlled trial. JAMA
2003;290(11):1459-1466
20. Leowattana W, Bhuripanyo K, Singhaviranon L, Akaniroj S, Mahanonda N, Samranthin M,
Pokum S. Roxithromycin in prevention of acute coronary syndrome associated with
Chlamydia pneumoniae infection: a randomized placebo controlled trial. J Med Assoc Thai
2001;84 (S3):S669-S675
21. Neumann F, Kastrati A, Miethke T, Pogatsa-Murray G, Mehilli J, Valina C, Jogethaei N, da
Costa CP, Wagner H, Schomig A. Treatment of Chlamydia pneumoniae infection with
roxithromycin and effect on neointima proliferation after coronary stent placement (ISAR-3): a
randomised, double-blind, placebo-controlled trial. Lancet 2001; 357(9274):2085-2089
22. Weiss SM, Robin PM, Gaydos CA, Cummings P, Patton DL, Schulhoff N, Shani J, Frankel R,
Penny K, Quinn TC, Hammerschlag MR, Schachter J. Failure to detect Chlamydia
pneumoniae in coronary atheromas of patients undergoing atherectomy. J Infect Dis
1996;173:957-962
23. Paterson DL, Hall J, Rasmussen SJ, Timms P. Failure to detect Chlamydia pneumoniae in
atherosclerotic plaques of Australian patients. Pathology 1998;30:169-172
24. Daus H, Ozbek C, Saage D, Scheller B, Schieffer H, Pfreundschuh M, Gause A. Lack of
evidence for a pathogenic role of Chlamydia pneumoniae and cytomegalovirus infection in
coronary atheroma formation. Cardiology 1998;90:83-88
25. Lindholt JS, Ostergaard L, Henneberg EW, Fasting H, Andersen P. Failure to demonstrate
Chlamydia pneumoniae in symptomatic abdominal aortic aneurysms by nested polymerase
chain reaction (PCR). Eur J Vasc Endovasc Surg 1998;15:161-164
26. Ursi D, Hoymans V, Altwegg M, Bosmans J, Van Marck E, Van Schil P, Vrints C, Goossens
H, Ieven M. Failure to detect Chlamydia pneumoniae (C.pn) DNA in human atherosclerosic
plaques or blood samples but demonstration of C.pn antigen in atherosclerotic plaques.
Abstracts of the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy,
Chicago, 2001;(L-2173) 479
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27. Berg HF, Maraha B, Scheffer GJ, Peeters MF, Kluytmans JA. Effect of clarithromycin on
inflammatory markers in patients with atherosclerosis. Clin Diagn Lab Immunol
2003;10(4):525-528
28. Berg HF, Tjhie JHT, Stobberingh EE, Peeters MF, van Keulen PHJ, Kluytmans JAJW. Effect
of clarithromycin on oropharyngeal and nasal flora: a double-blind placebo controlled study.
Clin Microbiol Infect 2003;9(S1):249
29. Anderson J.L., Muhlestein JB, Carlquist J, Allen A, Trehan S, Nielson C, Hall S, Brady J,
Egger M, Horne B, Lim T. Randomized secondary prevention trial of azithromycin in patients
with coronary artery disease and serological evidence for Chlamydia pneumoniae infection:
the azithromycin in coronary artery disease: Elimination of myocardial infection with
Chlamydia (ACADEMIC) study. Circulation 1999;99:1540-1547
86
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7
EMERGENCE AND PERSISTENCE OF MACROLIDE RESISTANCE IN
OROPHARYNGEAL FLORA AFTER CLARITHROMYCIN THERAPY:
A RANDOMISED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY
Berg HF, Tjhie JHT, Scheffer GJ, Peeters MF, van Keulen PHJ, Kluytmans JAJW,
Stobberingh EE
Submitted
CHAPTER 7
SUMMARY
To investigate the effect of clarithromycin slow-release on colonisation and the
development of resistance in oropharyngeal and nasal flora, a double-blind, randomised
placebo-controlled trial was performed with 8 weeks follow-up.
296 patients with documented coronary artery disease were randomised in the preoperative outpatient clinic to receive a daily dose of clarithromycin 500 mg, slow-release
SR (CL) or placebo tablets (PB) until the day of surgery. Nose and throat swabs were
taken before starting therapy, directly after end of therapy and 8 weeks later. Presence
and Minimal Inhibitory Concentration (MIC) were determined for oral Haemophilus
species and nasal Staphylococcus aureus. Quantitative culture on media with and
without macrolides was performed for the indigenous oropharyngeal flora. In addition,
genotypical analysis was performed on the macrolide resistant indigenous flora. Basic
patient characteristics were comparable in the two treatment groups. The average
number of tablets taken was 15 (SD=6.4). Oropharyngeal carriage of Haemophilus
species was not affected in either of the treatment groups. Nasal carriage of S. aureus,
however, was significantly reduced in the CL group (from 35.3% to 4.3%) compared to
the PB group (from 32.4% to 30.3%; p<0.0001; relative risk (RR), 7.0; 95% CI, 3.1-16.0).
Resistance to clarithromycin was present significantly more frequently after treatment in
the CL-group in Haemophilus species (p=0.007; RR, 1.6; 95% CI, 1.1-2.3); also, the
percentage of patients with resistance to macrolides in the indigenous flora after
treatment was significantly higher in the CL-group (31% to 69%; p<0.0001; RR, 1.9; 95%
CI, 1.4-2.5). This persisted for at least 8 weeks.
This study shows that besides the effective elimination of nasal carriage of S. aureus,
treatment with clarithromycin SR for approximately 2 weeks has a marked and sustained
effect on the development of resistance in the oropharyngeal flora for at least 8 weeks.
88
CLARITHROMYCIN AND EMERGING MACROLIDE RESISTANCE
INTRODUCTION
Antimicrobial agents are one of the most useful groups of therapeutic agents
available today. In fact it is the only group of therapeutic agents which has had a
measurable effect on overall mortality rates in the population1. Unfortunately, emergence
of resistance against many agents in almost all human pathogens is now widespread
and a cause of great concern for future therapeutic effectiveness2. Development of
resistance has been associated with the use of antimicrobial agents in general2,3.
In 1956, macrolides were introduced and presented as alternatives to penicillins,
especially for the treatment of infections due to Gram-positive micro-organisms4.
Currently, they are recommended as a first-line therapy for adults with communityacquired pneumonia5. Unfortunately, resistance to macrolides among Streptococcus
pneumoniae, the most common cause of respiratory tract infections, is increasing6,7.
There is little doubt that this is mainly the result of the increased use of macrolides.
Few papers have been published with regard to the effect of macrolides on the
oropharyngeal flora8-12. Moreover, no placebo-controlled randomised study has been
performed to study the extent of these effects.
The current study was performed to quantify the effect of clarithromycin on the
development of resistance in oropharyngeal and nasopharyngeal flora in individual
patients. This study was part of an ongoing intervention study in cardiothoracic surgery,
to study the effects of clarithromycin on the presence of Chlamydia pneumoniae in
cardiovascular tissue.
PATIENTS AND METHODS
Study population.
Between July 1999 and July 2001, patients with documented
coronary artery disease, who were scheduled for coronary artery bypass graft (CABG)
surgery, were enrolled in the study. Inclusion was carried out during a visit to the preoperative outpatient clinic at the department of cardio-thoracic surgery of the Amphia
Hospital in Breda, The Netherlands. Exclusion criteria included the following:
concomitant administration of terfenadine, rifabutin, cisapride or antipyrine; antibiotic
therapy with a macrolide, tetracycline, or quinolone within three months prior to inclusion
or during the study period. After giving informed consent, patients were enrolled in a
89
CHAPTER 7
double-blind, randomised, placebo-controlled study. From that point on, they started
taking the study medication until the day of surgery. The study medication consisted of
either a daily dose of slow-release clarithromycin 500 mg (CL) or a placebo (PB) tablet
(Clarithromycin SR and matching placebo tablets were obtained from Abbott
Laboratories, Abbott Laboratories Ltd, Queenborough, Kent, England ME11 5EL).
An independent pharmacist dispensed either CL or PB tablets according to a
computer generated randomisation table which stratified in groups of 10. The researcher
responsible for seeing the patients allocated the next available number on entry into the
trial, and each patient collected the corresponding tablets direct from the researcher. The
code was revealed to the researcher once recruitment, data collection, and laboratory
analyses were complete.
From each patient a nose swab and 2 throat swabs were obtained on inclusion
during the pre-operative outpatient visit (visit 1), and on the day of surgery just before the
operation a second nose swab and 2 throat swabs were obtained (visit 2). A third throat
swab was taken 8 weeks after surgery (visit 3). These swabs were stored in transport
medium (Transwab™ or a Venturi Transystem ®) at 4°C until further processing.
Microbiological analysis.
The nasal swabs were cultured on a blood agar
plate and incubated at 35°C for 48 hours. On days 1 and 2 the plates were examined for
the presence of Staphylococcus aureus. Putative colonies were identified using standard
identification methods13. S. aureus strains were deep-frozen in a bead storage system at
-80°C (Protect, Technical Service Consultants Limited, England) until further testing.
Throat swabs were cultured on blood agar plates containing 5µg/ml gentamicin and
chocolate agar plates supplied with a bacitracin tablet (Neo-sensitabs ®, Rosco,
Denmark). These plates were incubated at 35°C in the presence of 5% CO2 for 48
hours. On days 1 and 2 the throat culture plates were examined for the presence of
potential pathogenic micro-organisms, e.g. S. pneumoniae, Haemophilus spp., S.
pyogenes and Moraxella catharralis using standard identification methods14. All identified
strains were frozen at -80°C until further testing.
Sensitivity analysis. Phenotypical resistance was measured by determining the
minimal inhibitory concentration (MIC) for clarithromycin using E-test® (AB-Biodisk,
Sweden) according to the manufacturers guidelines. H. influenzae ATCC 49247 and S.
aureus ATCC 29213 were included as controls. MIC determination was performed by
reading at the point of complete inhibition of all growth, including micro-colonies, hazes
and isolated colonies using a plate microscope. Breakpoint for sensitive versus resistant
90
CLARITHROMYCIN AND EMERGING MACROLIDE RESISTANCE
were determined according to NCCLS guidelines (NCCLS MIC H. influenzae ≥ 32 ul/ml
is resistant).
Indigenous flora.
Culture and resistance detection for erythromycin of the
total indigenous oropharyngeal flora was performed for all three throat swabs. The
swabs were suspended in 1 ml 0.9% NaCl and inoculated onto blood agar plates
containing 0 and 1 mg/L erythromycin using a spiral plater (Eddy Jet, IUL instruments,
I.K.S., Leerdam, The Netherlands). Firstly, it was determined whether there were any
macrolide resistant flora by growth on the erythromycin-containing blood agar. Secondly,
the percentage of resistant flora was quantitatively determined by dividing the number of
colonies on erythromycin containing plates by the number of colonies on plates without
erythromycin (x 100%), to indicate the proportion of macrolide resistance among the total
oropharyngeal flora.
To study the mechanism of resistance, strains (viridans streptococci and
coagulase negative staphylococci) resistant to macrolides were studied for the presence
of genes by PCR, conferring resistance to macrolides as described previously by
Sutcliffe et al. and Jensen et al.15-17: erm B (target modification by a ribosomal mutation),
and mef A/E (macrolide efflux pump mechanism) genes for streptococci and erm A, erm
C (target modification by a ribosomal mutation) and msr (macrolide efflux pump
mechanism) genes for staphylococci. Some of these genes are known to occur on
common mobile genetic elements such as transposons, which facilitate the spread of
these resistance genes. The transposon family Tn916/Tn1545 is known to also harbour
erm genes18 and is common among Streptococci. In addition the presence of these
genes on Tn 916 / Tn 1545 was analysed by PCR19.
This study was approved by the local Medical Ethics Committee.
Statistical analysis. For statistical analysis all base-line characteristics were analysed
using the χ2-test for the distribution of categorical variables and the Student’s t test to
compare continuous variables. To assess differences in MIC values between the two
treatment groups and between variables with different standard deviations, a
comparison was made using the Mann-Whitney U-test. The related samples were
compared using a Wilcoxon Signed Ranks Test. To determine the correlation between
the duration of therapy and the development of resistance, linear regression analysis
was performed. Statistical significance was accepted when P<0.05.
91
CHAPTER 7
380 total eligible patients
Allocation
Enrolment
84 excluded
13 other surgical procedure
9 did not understand the study protocol
23 refused participation
14 used macrolide, quinolone or tetracycline
antibiotics ≤ 3 months prior to inclusion
14 participated in other trials
2 liver disease (elevated liver enzymes)
5 renal disease (elevated kreatinin)
4 allergy to macrolides
296 randomised and
screened for culture of
pathogens
148 placebo
109 clarithromycin
6 excluded from analysis
because of concomitant
use of other antibiotics
143 analysed
for pathogens
110 placebo
3 excluded from analysis
because of concomitant
use of other antibiotics
147 analysed
for pathogens
107 analysed for
indigenous flora
26 lost to
follow-up
Analysis
swab-3
Analysis
swab-1 / -2
Follow-up
148 clarithromycin
219 patients screened
for indigenous floraa
81 analysed for
indigenous flora
109 analysed for
indigenous flora
21 lost to
follow-up
88 analysed for
indigenous flora
Figure 1. Trial profile.
a
Screening of indigenous oropharyngeal flora was performed on a different lab and started on a later date
than the culture of pathogens
RESULTS
Figure 1 shows that a total of 296 patients were enrolled in the study and screened for
culture of oropharyngeal pathogens. Of these, 219 were also screened for indigenous
92
CLARITHROMYCIN AND EMERGING MACROLIDE RESISTANCE
oropharyngeal flora. After randomisation 148 patients received clarithromycin 500 mg
SR (CL group) and 148 placebo (PB group) (resp. 109 and 110 for patients screened for
indigenous oropharyngeal flora). Table 1 shows that the patient characteristics are well
balanced between the two treatment groups. No significant differences were found.
Six patients used other antibiotics concomitantly during the study period and
were excluded from further analysis. Nine patients were excluded from the analysis of
MIC determination for S. aureus, because the strains were not viable after storage.
Table 1. Patient characteristics.
Clarithromycin
(n=148)
Baseline characteristics
Mean
Age
64.6
Body mass index
27.6
Number of tablets used
14.9
Clarithromycin
(n=148)
Sex (male)
112
Smoker (currently)
28
Smoker in past
79
COPD
16
Diabetes Mellitus Type I 7
Type II 11
Other underlying disease
11
Immunosupressive therapy 5
Other antibiotics during 5
study period
SD
8.4
3.1
6.4
%
75.7
9.5
26.7
5.4
2.4
3.7
3.7
1.7
1.7
Placebo
(n=148)
Mean
63.3
28.3
14.8
Placebo
(n=148)
118
28
90
10
9
19
5
2
1
SD
8.9
3.7
6.0
%
79.7
9.5
30.4
3.4
3.0
6.4
1.7
0.7
0.3
SD = standard deviation
Pathogens.
The prevalence of S. pneumoniae, S. pyogenes and M. catharralis in throat
cultures was low. S. pneumoniae was found in five patients before treatment and in five,
other patients, after treatment. M. catharralis was found five times before treatment, and
in three other patients after treatment. S. pyogenes was found in two patients before
treatment, but none after. Before treatment, Haemophilus parainfluenza was the only
potential pathogen found consistently in the throat and S. aureus in the nose
(approximately 80% and 35% of patients respectively).
In table 2 the effect of clarithromycin on oropharyngeal carriage of H.
parainfluenza, and on nasal carriage of S. aureus is shown. Carriage of H. parainfluenza
was not significantly affected by clarithromycin therapy. Nasal carriage of S. aureus,
however, was significantly reduced in the CL group (from 35.3% to 4.3%) compared to
the PB group (from 32.4% to 30.3%, p<0.0001, RR: 7.0; 95% CI, 3.1 - 16.0).
93
CHAPTER 7
Table 2. Number of patients with nasal carriage of S. aureus and oropharyngeal carriage of
Haemophilus parainfluenza.
Clarithromycin %
Placebo
%
p-value
S. aureus
N=139
N=142
Nasal carriage
Before therapy
49
35.3
46
32.4
NS
After therapy
6
4.3
43
30.3
<0.0001
Clarithromycin
Haemophilus parainfluenza
N=143
Oropharyngeal carriage
Before therapy
117
After therapy
104
%
Placebo
%
N=147
81.8
72.7
117
110
79.6
74.8
NS
NS
NS= not significant
In Figure 2, the percentage of patients with clarithromycin-resistant H.
parainfluenza strains before and after therapy is shown for both groups. It shows a
significant increase in resistance in the CL-group and not in the PB-group (p=0.007; RR,
1.6; 95% CI, 1.1 - 2.3).
percentage of resistance
% 100
90
80
70
60
50
40
30
20
10
0
before
after
clarithromycin
placebo
Figure 2. Percentage of clarithromycin-resistant oropharyngeal H. parainfluenzae before and
after therapy in both treatment groups.
Statistical analysis was not performed on MICs of S. aureus due to the small
number of patients with S. aureus after treatment in the CL-group.
94
CLARITHROMYCIN AND EMERGING MACROLIDE RESISTANCE
Indigenous flora.
Throat swabs were obtained from 219 patients on visits 1 and 2. A third swab
was obtained from 169 of these 219 patients. Clarithromycin did not reduce the number
of CFUs/ml or the total amount of oral flora significantly.
Figure 3 shows the percentage of patients with macrolide-resistant oropharyngeal
flora for both the PB- and the CL-group at the three different points in time. In the CLgroup the percentage of patients with macrolide-resistant flora in the first swab was
identical to the placebo group (34%). Directly after treatment in the CL-group there was a
significant shift towards more patients with resistant oral flora (p<0.0001; RR 1.9; 95%
CI, 1.4 - 2.5), which was more or less the same in the third throat swab (p=0.003; RR
1.6; 95% CI, 1.2 - 2.1). The risk of developing resistance in a subgroup of patients
without detectable resistance before therapy was started was significantly higher in the
CL group compared to the PB group (p<0.0001; RR, 6.6; 95% CI, 3.2 - 13.5).
Percentage of patients with resistant flora
Clarithromycin
Placebo
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Swab1
Swab 2
Swab 3
Figure 3. Percentage of patients with macrolide-resistant gram-positive strains in clarithromycin
and placebo group.
Linear regression analysis showed that there was no significant relation between
the number of tablets taken and the prevalence of resistance in the CL-group.
95
CHAPTER 7
Quantitative culture was performed to determine in each individual patient with
resistant flora which percentage of the total oral flora was resistant. In the pre-treatment
situation (visit 1), the mean percentage of the total flora which was resistant in the CLgroup and PB-group was 3.4% and 2.3% respectively. Immediately after treatment (visit
2) the mean percentage of the total flora which was resistant rose to 21.1 % in the CLgroup (versus 1.1% in the PB group, p<0.0001). This increase was still present 8 weeks
later (11.4% versus 2·6%, p<0.0001).
Resistance genes present before treatment, were not significantly different
between the treatment groups. However, after treatment a significant rise in erm B was
seen in the streptococci (p<0.0001; RR, 3.5; 95% CI, 2.0-6.1) as well as a significant rise
in erm C in staphylococci (p=0.002; RR, 4.5; 95% CI, 1.6-12.8) in the CL group
compared to the PB group. This difference was still present 8 weeks after treatment.
More than 90% of the tested macrolide-resistant streptococci contained both macrolideresistance genes and Tn916/Tn1545 fragments.
DISCUSSION
This is the first double-blind, placebo-controlled randomised study that quantifies the
effect of an antibiotic on the development of resistance in the oropharyngeal flora. It
shows that administration of a daily dose of 500 mg of slow-release clarithromycin for 2
weeks has major effects that are still present 8 weeks later.
Before patients were included in the study, macrolide resistance was already
frequently present. About 35% of patients carried H. parainfluenzae strains that were
clarithromycin-resistant and about 35% of the patients had some macrolide-resistant
indigenous flora. This despite the fact that patients who had recently (≤ 3 months before
inclusion) been treated with macrolides had been excluded from the study. Whether this
rate of resistance is due to macrolide therapy in the more distant past (>3 months) or
whether it is the normal rate in the oropharyngeal flora in this population is not clear.
Immediately after treatment the percentage of patients with macrolide-resistant
flora in the CL-group more or less doubled: this was found for both macrolide resistant H.
parainfluenzae as for macrolide-resistant indigenous oropharyngeal flora. This effect on
the indigenous flora was even greater if only the group of patients were taken into
consideration that did not carry macrolide-resistant flora. The relative risk of developing
96
CLARITHROMYCIN AND EMERGING MACROLIDE RESISTANCE
macrolide resistance in the indigenous flora directly after treatment in this group was 6.6
(95% confidence Interval: 3.2-13.5).
Unexpectedly, this effect was more or less the same 8 weeks later. Other
investigators found that emergence of resistance after clarithromycin therapy had
disappeared after 6 weeks20. In that study another group of patients was treated with
azithromycin. In this group the resistance rate after 6 weeks was even higher than after
one week. The authors speculate that this difference is caused by the extremely long
elimination half-life of azithromycin compared to clarithromycin, which leads to subinhibitory concentrations for several weeks. In our study clarithromycin was used in a
slow-release form, with a prolonged elimination half-life for once daily dosing (5.3 hrs,
compared to 2.7 hrs for the normal formula). Although this half-life is not as long as that
of azithromycin (up to 72 hrs) it is possible that the prolonged effect of clarithromycin SR
on resistance is partly caused by this prolonged half-life. A big difference however is that
we were studying patients with gram-positive resistant indigenous flora and H.
parainfluenzae, whereas Kastner and Guggenbichler were studying specific pathogens
that are not considered to be part of the normal oropharyngeal flora, such as
Enterobacter spp., Klebsiella spp. and Pseudomonas spp.
Since we anticipated that the effect of CL after 8 weeks would have largely
disappeared, we did not plan a follow-up beyond that point. Considering our findings, a
further follow-up would have been useful. Nevertheless, the conclusion is justified that a
significant and sustained effect on resistance is produced by clarithromycin SR for at
least 8 weeks.
Furthermore, the percentage of resistance in the total flora in each individual
patient was quantified and very low percentages of resistant flora could be detected with
the method used. To check for the effect of the duration of treatment, an analysis that
compared the duration of treatment with the percentage of resistant flora was performed.
No association was found. This finding supports the hypothesis that development of
resistance was mainly caused by selecting macrolide-resistant isolates, because they
were already detected after a few tablets. This was also suggested by Leach et al. who
concluded that the selective effect of macrolides allowed the growth and transmission of
pre-existing macrolide-resistant strains21. The favorable circumstances for macrolideresistant flora is illustrated by the fact that more patients had high proportions of the total
flora being resistant directly after therapy, compared to the pre-treatment situation.
Although these high proportions decreased slightly after 8 weeks, the percentage of
97
CHAPTER 7
patients with detectable resistance was still as high as immediately after therapy. This is
particularly worrying as our results indicate that the resistance genes are associated with
mobile genetic elements, which facilitate the spread of these genes to more pathogenic
flora as well.
Another clear effect of clarithromycin was the effect on the distribution of the
different resistance genes in the oropharyngeal streptococci and staphylococci. A
significant rise of the erm B gene (which encodes for target modification) was detected in
streptococci as well as a significant rise of the erm C gene in staphylococci. Target
modification due to a mutation of 23S rRNA or ribosomal protein, was the most
frequently found resistance mechanism in the oropharyngeal flora after CL-therapy,
which was also found by other investigators 15-17.
In conclusion, there was substantial development of resistance that was longlasting. These findings deserve serious consideration when macrolides are used,
especially when this is for indications other than infectious diseases. For example,
erythromycin is known for the treatment of gastroparesis as a gastrointestinal prokinetic
agent
administered
orally
or
intravenously22.
Also
anti-inflammatory
and
immunomodulatory properties are mentioned in various pulmonary situations such as
diffuse panbronchiolitis (DPB)23. Given the major effect on the development of resistance
in the indigenous and pathogenic flora, one should reconsider prescribing macrolides
and weigh these results against the evidence of the suggested gain.
Finally, nasal carriage of S. aureus was significantly decreased. In general,
systemic antibiotic treatment affects the balance between commensal and pathogenic
microflora, but very few are capable of decreasing the carriage of S. aureus. A study by
Yu et al., for example, showed a decrease in nasal carriage after administration of
rifampicin24. Our study showed that clarithromycin SR is capable of decreasing
staphylococcal nasal carriage from 35.3% to 4.3%, which is comparable to the activity of
mupirocin nasal ointment25. Eradication of S. aureus is important, because elimination of
nasal carriage has been found to reduce infection rates and the severe consequences of
infection in patients at risk26. However, because of the development of resistance, the
use of macrolides for this indication is questionable.
In conclusion, our study documents that administration of clarithromycin SR
results in a significant elimination of S. aureus nasal carriage, but on the other hand
induces a rapid and prolonged increase in macrolide resistance in oropharyngeal flora.
The macrolide-resistance genes are similar to those usually detected in pathogenic flora.
98
CLARITHROMYCIN AND EMERGING MACROLIDE RESISTANCE
This study provides indisputable evidence that treatment with clarithromycin results in a
major effect on the sensitivity of the oropharyngeal flora to macrolides for at least 8
weeks.
Acknowledgment. We thank the laboratory for Clinical Micobiology of the Amphia
hospital and University Medical Centre Maastricht for their technical assistance.
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CHAPTER 7
REFERENCES
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2. Neu HC. The crisis in antibiotic resistance. Science 1992;257:1064-1073
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101
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SUMMARY AND CONCLUSIONS
8
CHAPTER 8
SUMMARY and CONCLUSIONS
In 1988 observational evidence for a possible association of C. pneumoniae with
coronary artery disease (CAD) was first described1. Since then, efforts have been made
to determine the pathogenic role of C. pneumoniae in atherosclerosis. Several types of
evidence have suggested this role, derived from seroepidemiological studies, direct
identification of C. pneumoniae in atherosclerotic lesions, in vitro experimental studies,
animal model experiments and antibiotic treatment trials in patients with CAD.
Initially, seroepidemiological studies indicated that patients with CAD had a high
anti-C. pneumoniae antibody titer, although more recent studies have challenged this2,3.
It is uncertain whether antibodies to C. pneumoniae reflect active infection, past
infection, chronic infection, or antigenic cross-reactivity. The conflicting results between
serological studies are possibly due to lack of uniformity in methods used (different
classes of antibodies tested, different methods, different cutoff points, cross-reaction to
non-C. pneumoniae antigens4,5,6). In several retrospective studies no adjustment for
confounding factors, such as important CAD risk factors was done. Finally, the high
prevalence of C. pneumoniae exposure in the population makes it difficult to identify true
differences between those who are actively infected and those who are not.
In addition to serology, data from basic laboratory studies have provided
evidence for the presence of C. pneumoniae in atherosclerotic lesions. Direct detection
of organisms by immunohistochemistry (IHC), electron microscopy, in-situ hybridization,
or amplification of chlamydial DNA by polymerase chain reaction (PCR) has shown that
C. pneumoniae can be found in atheromas. Subsequently, viable organisms have been
detected by amplification of mRNA transcripts from atheromas or have been isolated by
culture from atherosclerotic tissue. Chapter 2 describes a pilot study that was conducted
to identify the presence of C. pneumoniae in Dutch patients from our region. C.
pneumoniae DNA was detected by PCR in 22% (10/45) of the atherosclerotic samples
and in 10% (5/50) of non-atherosclerotic samples. IHC staining was positive for C.
pneumoniae in 60% (25/42) of the atherosclerotic specimens and in 9% (4/46) of the
non-atherosclerotic specimens. The correlation between the detection of C. pneumoniae
by PCR and IHC was 0.35 (p=0.003) for atherosclerotic specimens. A poor correlation of
0.1 (p>0.05) was found between these detection techniques in non-atherosclerotic
specimens. Overall, these study results support the finding of a high prevalence of C.
pneumoniae in atherosclerotic lesions, when detected by IHC or PCR. Data collected
104
SUMMARY AND CONCLUSIONS
from 43 studies by Kalayoglu et al, indicated a high prevalence of C. pneumoniae (46%
of 1852) in atheromatous tissue specimens, but a low prevalence of less than 1% in
healthy arteries7.
In a second study, we determined the correlation between C. pneumoniae
serology and the presence of C. pneumoniae as detected by PCR and IHC. As
described in chapter 2, this correlation appeared very poor. Several other studies have
reported the same: C. pneumoniae serology frequently does not correlate with the
identification of the organism by PCR or IHC8. The reliability of anti-chlamydial IgG titers
for predicting the intravascular presence of the pathogen can therefore be questioned2,3.
A limitation of chapter 2 is that we did not use established guidelines for the
interpretation of agreement between tests9. According to these guidelines, the
agreement between PCR, IHC, and serology found in our study and in most studies
reviewed in chapter 2 is just fair. In contrast, Juvonen and Shor, found a good correlation
between the different diagnostic techniques10,11. This latter reanalysis weakens the
conclusions that we have drawn in chapter 2 and stresses the importance of
interpretation of the detection methods of C. pneumoniae, and its limitations.
Increasing interest in the potential link between atherosclerosis and C.
pneumoniae infection has led to many studies involving C. pneumoniae detection by
PCR. Because PCR was potentially an important tool for further studies in this field,
many investigators have used this method, but the results reported so far are
contradictory and confusing. A considerable variation in the detection rate of C.
pneumoniae, ranging from 0 % to 100%, has been reported by different investigators.
The detection varies depending on the methods used but also when independent
investigators use similar methods. A multi-centre study showed major interlaboratory
differences in the detection rate of C. pneumoniae in endarterectomy specimens12.
Obviously, standardization of the detection methods is needed for optimal C.
pneumoniae identification.
In order to efficiently extract DNA from vascular tissue and establish an optimal
sensitivity of C. pneumoniae identification by PCR, four procedures for C. pneumoniae
DNA extraction from vascular tissue were compared in chapter 3. Evaluation was based
on the yield of recovered DNA, using PCR to detect C. pneumoniae in vascular tissue.
The QIAamp DNA MiniKit was found to be a useful and most efficient extraction method,
compared to the NucliSens Kit, the buffer-saturated phenol method and the Geneclean II
Kit. It also had a short handling time, but was more expensive. It should be mentioned
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CHAPTER 8
that spiked materials and non-spiked atheroma specimens are similar but not absolutely
identical12, and that it is not known whether the capacity of these different methods to
extract C. pneumoniae DNA from spiked materials is the same as the capacity to extract
DNA from patient materials. In our hands, the QIAamp DNA MiniKit was the most
efficient method, therefore this method for DNA extraction was used in all further studies
described in this thesis.
Since initial seroepidemiologic studies and detection studies showed a clear
association between C. pneumoniae and augmented atherogenesis, antibiotic
intervention trials were initiated. The underlying rationale is that antibiotics effective
against C. pneumoniae may lower the risk for subsequent cardiac events in patients with
CAD, by eradicating the organism from plaques, thus stabilizing the lesions13. Gupta et
al. were the first to find a significant risk reduction of cardiovascular events after a short
(3- to 6-day) course of azithromycin treatment in 220 male survivors of myocardial
infarction with anti-C. pneumoniae antibody titers14. These promising results prompted
us to conduct a prospective, randomized, placebo-controlled intervention trial in patients
with severe atherosclerosis. The objective of this trial was to examine the effects of
clarithromycin in patients with documented coronary artery disease. Patients who were
scheduled for coronary artery bypass graft surgery (CABG) were included in the
intervention trial. All patients were randomly assigned to receive either clarithromycin
(500 mg SR) or placebo until the day of surgery.
In chapter 4, the effect of treatment with clarithromycin on the presence of C.
pneumoniae in vascular tissue and on antibodies against Chlamydia in serum was
assessed. Despite the use of different detection methods, no evidence for the presence
of viable C. pneumoniae in vascular tissue of CAD patients could be found.
Clarithromycin treatment of CAD patients had neither an effect on the presence of C.
pneumoniae antigens in vascular tissue nor an effect on circulating Chlamydia IgG
antibody titers.
Vascular specimens were tested by an automated real-time PCR, and an industrydeveloped LCx-C. pneumoniae PCR. However, with both methods we failed to detect C.
pneumoniae DNA in any specimen. This indicates that there is no evidence of acute C.
pneumoniae infection in vascular tissue of CAD patients. Other possible explanations for
the negative results of the PCR assays could be the low DNA concentration in the test
samples and the patchy distribution of C. pneumoniae DNA in vascular tissue15.
106
SUMMARY AND CONCLUSIONS
In the present study an automated real-time PCR was used, which combines
amplification, hybridization and quantitative product detection. Lately, many research
groups have switched to this new, real-time based PCR technology, which is less prone
to contamination16-18. In earlier studies conventional PCR assays were used. It is
noteworthy that the rate of positivity of the PCR at our laboratory became lower, as more
laboratory experience was gained in the molecular assays of C. pneumoniae. Also,
multicenter studies demonstrated inter- en intra-laboratory discrepancies in the detection
of C. pneumoniae. Altogether, these findings imply that the results generated by
conventional PCR assays, including those from our studies, are probably biased by
inexperience, choice of DNA polymerase, as well as contamination and crossreactivity8,19.
Whereas (real-time and industry developed) PCR assays were negative in all
specimens, we could detect C. pneumoniae MOMP antigen in the majority of the
specimens by IHC (73.8% in the clarithromycin group vs. 77.0% in the placebo group),
but Chlamydia LPS antigen was found in only one specimen. The abundance of MOMP
in
complete
absence
of
C.
pneumoniae
DNA
is
also
described
by other
investigators8,20,21. It is possible that MOMP antigens of C. pneumoniae might persist for
a longer period of time, while C. pneumoniae DNA and LPS may degrade more rapidly in
vascular tissue. Clarithromycin treatment did not have any influence on MOMP antigen
detection in the atherosclerotic lesions, which further corroborates the notion that this
antigen can remain unaltered for longer periods. This has also been demonstrated in an
animal experiment in which antibiotic treatment with azithromycin did not lead to
elimination of chlamydial antigen from vascular specimens of rabbits infected with C.
pneumoniae22. Also, in a mice model, azithromycin treatment did not affect the presence
of C. pneumoniae in the aorta, lung, or spleen23. IHC may allow localization of the
organisms in specific cells and areas. However, protocols for performance and
interpretation of IHC are not standardized. This technique requires subjective reading
and its interpretation remains difficult because of background and nonspecific staining8.
The results concerning the high detection rate of C. pneumoniae MOMP antigen
and the low detection rate of C. pneumoniae DNA might be partly explained by the
biology of chlamydiae. Infections with chlamydiae (e.g. C. trachomatis) are characterized
by alternate phases of activity and latency followed by fibrosis and scarring24. In
advanced infections, chlamydiae are no longer viable. The low prevalence or absence of
C. pneumoniae DNA and LPS in vascular specimens, and the presence of the MOMP
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CHAPTER 8
antigen supports the theory that there are no viable micro-organisms present in
advanced atherosclerosis. According to this hypothesis, effects of antibiotic treatment on
the elimination of Chlamydia from the lesions should be studied in patients in the initial
stage of atherosclerosis24.
Apart from an established chemotherapeutic potential, another therapeutic
potential of macrolide antibiotics might be an anti-inflammatory effect25. Because
atheromas are characterized by lesions that contain abundant immune cells, particularly
macrophages and T cells, atherosclerosis can be regarded as an inflammatory disease.
In addition, C. pneumoniae, as viable or dead organism, may cause a nidus for chronic
inflammation, in which macrolide antibiotics might work as anti-inflammatory drugs and
thus reduce cardiovascular risk. Since there was much speculation about the antiinflammatory potential of macrolides in patients with CAD, the effect of clarithromycin on
inflammatory markers was studied (chapter 5). Treatment with clarithromycin for on
average 16 days did not significantly alter the inflammatory markers C-reactive protein
[CRP], interleukin [IL]-2R, IL-6, IL-8, and tumour necrosis factor (TNF)-α), nor on the day
of surgery, nor 8 weeks later. The anti-inflammatory potential of clarithromycin in these
patients was therefore considered as negligible. In an intervention study with
roxithromycin in 202 patient with unstable angina or non-Q-wave myocardial infarction
Gurfinkel et al. found a larger decrease of CRP in the roxithromycin treatment group
after 31 days than in the placebo group (p=0.03). However, the CRP base-line levels
were not associated with major ischemic events26. Anderson et al. found a reduction in a
composite of inflammatory markers of 302 patients with antibody titers against C.
pneumoniae after azithromycin treatment27. Although in both studies these results were
statistically significant, the data were based on minor clinical changes. A clear antiinflammatory effect of macrolides in patients with CAD was not shown.
Since in a few small intervention studies it was found that antibiotic treatment
might reduce cardiovascular risk in patients with CAD14,28, we performed a follow-up
analysis of our cohort, to study whether treatment with clarithromycin would prevent
future cardiovascular events and mortality. A total of 473 patients who were scheduled
for CABG surgery were finally included in the intervention trial, described in chapter 6.
Patients were treated for an average of 16 days. Follow-up at 2 years could be achieved
in 424 out of 473 patients. Total mortality was almost equal for both treatment groups
(p=0.81). Also, there were no significant differences in cardiovascular event rates during
the follow-up period (p=0.86). Treatment with clarithromycin in patients scheduled for
108
SUMMARY AND CONCLUSIONS
cardiac surgery did not reduce the subsequent occurrence of cardiovascular events or
mortality during a 2-year follow-up. A review of all randomised-controlled trials until now
in patients with established coronary artery disease demonstrated that the majority of
randomised-controlled trials did not show any beneficial effect in outcome after a course
of macrolides in these patients. The positive results of small trials14,29,30 were not
confirmed in larger trials, including ours. One factor might help explain this phenomenon.
Smaller trials may have been conducted in more selected patient populations, which
may indicate that certain groups of individuals might benefit from antibiotic treatment. In
the WIZARD trial, analysis within subpopulations showed trends toward a favorable
effect of antibiotic therapy in men who smoke or who have diabetes or
hypercholesterolemia. In the subgroup of patients who had diabetes and smoked the
event rate was 14.6% for those receiving azithromycin compared to 53% for those
receiving placebo31. In a subgroup analysis of the ISAR-3 trial, patients with the highest
titres of C. pneumoniae antibodies had a reduced rate of restenosis if given
roxithromycin compared to placebo32. In our study results of treatment with
clarithromycin did not differ for patients who had diabetes or those who had not, and
those who were smoking, or were not. Therefore, no recommendation for the use of
antibiotic therapy for the secondary prevention of atherosclerosis could be made.
The use of antibiotics is associated with the emergence of resistance against
these agents in almost all human pathogens. This is a widespread problem and cause of
great concern for future therapeutic effectiveness33,34. Therefore, use of macrolide
antibiotics such as clarithromycin deserves serious thought when they are used for other
purposes than infectious diseases such as CAD. Chapter 7 documents the effect of
clarithromycin on the development of resistance in oropharyngeal and nasopharyngeal
flora in individual patients, as measured by agar dilution (MIC) and detection of
resistance genes (MLS) by PCR. Treatment with clarithromycin for 16 days caused a
major effect on the development of resistance in the oropharyngeal flora, which lasted
for at least 8 weeks. Immediately after treatment the percentage of patients with
macrolide-resistant flora in the CL-group more or less doubled (p<0.0001). Furthermore,
nasal carriage of S. aureus significantly decreased. Because resistance to macrolides is
increasing35,36, mainly due to the increased use, prescription of macrolides like
clarithromycin, should be restricted to clear indications.
Apart from patient-related studies, further evidence for the C. pneumoniaeatherosclerotic link came from animal model experiments in an attempt to fulfill Koch’s
109
CHAPTER 8
postulates. A problem is that these models are not completely comparable to the disease
in humans, since animals do not develop atherosclerosis naturally. Therefore animals
prone to atherosclerosis due to hyperlipemia are used (ApoE mice, low density
lipoprotein receptor mice, or New Zealand white rabbits). Introduction of C. pneumoniae
in the respiratory tract simulates the portal of entry in human infection. In these studies it
was established that C. pneumoniae has particular tropism for the vasculature and the
capacity to induce inflammation, and that it can initiate or promote lesion development in
hyperlipemic animals with atherosclerosis37-41. Hu et al, however, found that this
accelerating effect was correlated to the serum cholesterol level of the animals
38
.
Moreover, compelling evidence for the importance of the hyperlipidaemia and the less
significant role of Chlamydia, came from severely hyperlipidemic animals that developed
atherosclerosis in germ-free conditions42. In addition, several infectious agents other
than C. pneumoniae were shown to be able to induce similar atherosclerotic changes in
hyperlipidemic animals; therefore there does not seem to be a major atherogenic role for
C. pneumoniae37,43,44. Also more recent studies have failed to demonstrate a role for C.
pneumoniae in atherogenesis45,46. Further studies have tested the effects of antibiotic
treatment in modifying experimental atherogenesis in Chlamydia-infected animals.
Muhlestein et al first reported that azithromycin prevented accelerated atherosclerosis in
hyperlipidemic rabbits infected with C. pneumoniae22. More recent observations showed
that antibiotic treatment was more effective if initiated early after experimental infection
(< 1 week), which implies that antibiotic treatment has no effect in chronic infections23,47.
In conclusion, this thesis challenges an active role for C. pneumoniae in
atherosclerosis. The efforts to demonstrate the presence of C. pneumoniae in vascular
specimens by several detection techniques have yielded controversial results.
Unfortunately, available diagnostic methods like PCR and IHC require cautious
interpretation and standardization to detect or monitor acute, chronic or persistent C.
pneumoniae infection. Despite the use of several different detection techniques we were
not able to demonstrate the presence of an active infection with (viable) C. pneumoniae.
The clinical data of our randomized controlled trial do not support use of antibiotics for
prevention of cardiovascular events in patients with CAD. Eradication of C. pneumoniae
could not be demonstrated. An additional anti-inflammatory effect was not shown. Even
a two years follow-up period did not reveal any difference in the occurrence of
cardiovascular events in patients treated with clarithromycin or placebo. Moreover, a
110
SUMMARY AND CONCLUSIONS
significant and sustained resistance to macrolide antibiotics was assessed after a
relatively short-course of clarithromycin.
We must conclude that the true association between Chlamydia pneumoniae and
atherosclerosis is still not clear, but we consider a causal role of this micororganism
highly unlikely because of the complexity and multifactorial nature of atherosclerosis.
111
CHAPTER 8
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MR, Cook TD; Investigators in the WIZARD Study. Azithromycin for the secondary prevention
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Costa CP, Wagner H, Schomig A. Treatment of Chlamydia pneumoniae infection with
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Hermanowski-Vosatka A, Bergstrom JD, Sparrow CP, Detmers PA, Chao YS. Infectious
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Atherosclerosis in apoE knockout mice infected with multiple pathogens. J Infect Dis
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infection does not induce or modify atherosclerosis in mice. Circulation 2001;103(23):28342838
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Puolakkainen M, Penttila I, Saikku P. Chlamydia pneumoniae does not increase
atherosclerosis in the aortic root of apolipoprotein E-deficient mice. Arterioscler Thromb Vasc
Biol 2001;21(4):578-584
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atherosclerotic lesions after Chlamydia pneumoniae infection in a rabbit model. Antimicrob
Agents Chemother. 2002;46(8):2321-2326
115
SAMENVATTING EN CONCLUSIES
SAMENVATTING EN CONCLUSIES
In 1988 werd een mogelijke associatie tussen C. pneumoniae en coronaire arterieel
vaatlijden (CAV) voor het eerst experimenteel aangetoond. Sindsdien zijn pogingen
ondernomen om de pathogene rol van C. pneumoniae in atherosclerose te bepalen.
Verschillende vormen van bewijs hebben deze rol gesuggereerd, voortkomend uit
seroepidemiologische
studies,
de
directe
identificatie
van
C.
pneumoniae
in
atherosclerotische laesies, in-vitro experimenten, diermodel experimenten en antibiotica
interventie studies in patiënten met CAV.
Aanvankelijk bleek uit seroepidemiologische studies dat patiënten met CAV hoge
anti-C. pneumoniae antistoftiters hadden, maar meer recente studies betwijfelen dit. Het
is niet zeker of antistoffen tegen C. pneumoniae een actieve, chronische, in het verleden
doorgemaakte infectie of antigene kruisreactiviteit weerspiegelen. De tegenstrijdige
resultaten tussen serologische studies zijn mogelijk gevolg van een gebrek aan
uniformiteit tussen de gebruikte methoden (verschillende classen van geteste
antistoffen, verschillende methoden, verschillende grenswaarden, kruisreactiviteit tegen
niet-C. pneumoniae antigenen). Tevens werd in verschillende retrospectieve studies niet
gecorrigeerd voor beïnvloedende factoren. Een bijkomend probleem is dat het moeilijk is
om duidelijk onderscheid te maken tussen actief geïnfecteerden en niet actief
geïnfecteerden.
Naast studies gebaseerd op serologie leverden algemene laboratorium studies
bewijs voor de aanwezigheid van C. pneumoniae in atherosclerotische laesies. Directe
detectie van de organismen door middel van immunohistochemie (IHC), electronen
microscopie, in-situ hybridisatie of amplificatie van chlamydia DNA door middel van
polymerase ketting reactie (PCR) hebben de aanwezigheid van C. pneumoniae in
atheromen aan het licht gebracht. Vervolgens zijn levende organismen gedetecteerd
door amplificatie van mRNA transcripten uit atheromen of geïsoleerd door het kweken
van delende organismen uit atherosclerotisch weefsel. In Hoofdstuk 2 beschrijven wij
een pilot studie naar de detectie van C. pneumoniae. C. pneumoniae DNA werd
gedetecteerd door middel van PCR in 22% (10/45) van de atherosclerotische weefsels
en in 0% (5/50) van de niet-atherosclerotische weefsels. IHC kleuring was positief voor
C. pneumoniae in 60% (25/42) van de atherosclerotische monsters en in 9% (4/46) van
de niet-atherosclerotische weefsels. De correlatie tussen de detectie van C. pneumoniae
door middel van PCR en IHC was 0,35 (p=0,003) voor atherosclerotische weefsels. Een
118
SAMENVATTING EN CONCLUSIES
zwakke correlatie van 0,1 (p>0,05) werd gevonden tussen deze detectie technieken in
niet-atherosclerotische weefsels. Over het algemeen ondersteunen de studie resultaten
de bevinding van een hoge prevalentie van C. pneumoniae in atherosclerotische laesies
wanneer deze aangetoond wordt met behulp van IHC of PCR. Verzamelde data van 43
studies door Kalayoglu et al toonden een hoge prevalentie van C. pneumoniae (46% van
1852) in atheromateuze weefsels, maar minder dan 1% in gezonde arteriën.
Daartegenover vinden wij (hoofdstuk 2) geen correlatie tussen C. pneumoniae serologie
en detectie resultaten van zowel PCR als IHC. Verschillende andere studies kwamen tot
dezelfde bevinding dat C. pneumoniae serologie vaak niet correleerde met de
identificatie van het organisme door PCR of IHC. De betrouwbaarheid van antichlamydia IgG titers in het voorspellen van de intravasculaire aanwezigheid van het
pathogeen mag daarom worden betwijfeld. Een beperking van de studie die beschreven
wordt in hoofdstuk 2 is dat we de richtlijnen voor de interpretatie van de correlatie (lees
overeenkomst) tussen de test methodes niet hebben gebruikt. Volgens deze richtlijnen is
de overeenkomst tussen PCR en IHC in onze studie en de meeste gereviewde studies
uit hoofdstuk 2 redelijk. Een goede overeenkomst werd alleen gevonden door de
Juvonen en Shor et al.. Deze heranalyse zwakt de conclusies uit hoofdstuk 2 enigszins
af en benadrukt het belang van de interpretatie van de detectie methoden.
Toenemende interesse in de potentiele link tussen atherosclerose en C.
pneumoniae infectie heeft geleid tot vele studies waarbij C. pneumoniae gedetecteerd
wordt door middel van PCR. Omdat PCR potentiëel een belangrijk hulpmiddel is voor
aanvullende studies op dit gebied hebben veel onderzoekers deze methode gebruikt. De
resultaten zijn echter tegenstrijdig en verwarrend. Een aanzienlijke variatie in de C.
pneumoniae detectie werd gerapporteerd door diverse onderzoekers, variërend van 0%
tot 100%. De detectie variëerde tussen de diverse gebruikte methoden, maar ook tussen
verschillende onafhankelijke onderzoekers die vergelijkbare methodes gebruikten. Een
multi-centre studie toonde aanzienlijke verschillen in het detectie percentage van C.
pneumoniae in endartherectomie weefsels tussen laboratoria onderling. Het is duidelijk
dat voor optimale detectie van C. pneumoniae standaardisatie van de detectie methoden
een absolute voorwaarde is.
Om de meest efficiente methode te kiezen voor DNA extractie uit vaatwand
weefsel en een optimale gevoeligheid te bewerkstelligen voor C. pneumoniae detectie
door PCR, werden eerst vier C. pneumoniae DNA extractie methoden met elkaar
vergeleken (hoofdstuk 3). De evaluatie werd gebaseerd op de hoeveelheid vrijgekomen
119
DNA na extractie uit weefsel met verschillende concentraties toegevoegde C.
pneumoniae, waarbij PCR gebruikt werd om C. pneumoniae te detecteren in vaatwand
weefsel. De QIAamp DNA MiniKit bleek een nuttige en meest efficiente extractie
methode, vergeleken met de NucliSens Kit, de verzadigde-buffer phenol methode en de
Geneclean II Kit. Het had tevens een korte gebruikers tijd, maar was iets duurder. Het
moet benadrukt worden dat materialen waar DNA aan toegevoegd is vergelijkbaar zijn
met materialen waarbij dit niet is toegevoegd, maar zeker niet identiek zijn. Het is niet
bekend of C. pneumoniae DNA extractie uit materialen waar het van te voren kunstmatig
aan toegevoegd is even efficiënt verloopt als de extractie van C. pneumoniae DNA uit
natuurlijk geïnfecteerd patiënten materiaal. Omdat de QIAamp DNA MiniKit in onze
handen het meest efficiënt was, is deze DNA extractie methode verder gebruikt in alle
studies beschreven in dit proefschrift.
Sinds de veelbelovende resultaten van de eerste seroepidemiologische en
detectie studies, waarbij het verband tussen C. pneumoniae en toenemende
atherogenese werd gelegd, zijn antibiotica interventie studies geïnitiëerd. De theorie is
dat antibiotica die effectief zijn tegen C. pneumoniae het risico op cardiovasculaire
incidenten bij patiënten met CAV kunnen verminderen, doordat het organisme uit de
plaque wordt geëradiceerd en daarmee de plaque stabiliseert. Gupta et al. waren de
eersten die een significante afname van het risico in cardiovasculaire incidenten vonden,
na een korte behandeling (3 of 6 dagen) met azithromycine. Deze studie werd
uitgevoerd bij 220 mannelijke overlevenden van een myocard infarct met anti-C.
pneumoniae antistoftiters. Deze veelbelovende resultaten vormden de aanleiding voor
een prospectieve, gerandomiseerde, placebo-gecontroleerde interventie studie bij
patiënten met ernstige atherosclerose in ons centrum. Het doel van deze hoofdstudie
was de effecten van clarithromycine in patiënten met gedocumenteerd coronair
vaatlijden te onderzoeken. Patiënten die op de wachtlijst stonden voor coronair arteriële
bypass chirurgie (CABG) werden geïncludeerd. Alle patiënten werden gerandomiseerd
om hetzij met clarithromycine (500 mg SR) of met placebo behandeld te worden tot de
dag van chirurgie.
In hoofdstuk 4 beschrijven wij de studie waarin het effect van behandeling met
clarithromycine op de aanwezigheid van C. pneumoniae in vaatwand weefsel en op
antistoffen tegen Chlamydia in serum onderzocht werd. Ondanks het gebruik van
diverse detectie methoden, werd er geen bewijs gevonden voor de aanwezigheid van
levende C. pneumoniae in vaatwand weefsel van patiënten met CAV. Clarithromycine
120
SAMENVATTING EN CONCLUSIES
behandeling van patiënten met CAV had geen effect op de aanwezigheid van C.
pneumoniae antigenen in vaatwand weefsel, noch een effect op circulerende Chlamydia
IgG antistof titers. Vaatwand weefsels werden onderzocht met een geautomatiseerde
real-time PCR en een door de industrie ontwikkelde LCx-C. pneumoniae PCR. Met geen
van beide methodes konden wij echter C. pneumoniae DNA aantonen in welk van de
vaatwand weefsels dan ook. Deze studie levert dan ook geen bewijs voor de
aanwezigheid van een acute C. pneumoniae infectie in vaatwandweefsel van patiënten
met CAV. Andere verklaringen voor de negatieve resultaten van deze PCR testen zou
de lage DNA concentratie kunnen zijn in de geteste monsters en de vlekkerige verdeling
van C. pneumoniae DNA in vaatwand weefsel.
In de huidige studie werd een geautomatiseerde real-time PCR gebruikt, die
amplificatie combineert met hybridisatie en kwantitatieve product detectie. De laatste tijd
zijn veel onderzoeksgroepen overgeschakeld op deze nieuwe, op real-time gebaseerde
PCR technologie, die minder vatbaar is voor contaminatie. In eerdere studies werden
conventionele PCR methodes gebruikt. Het is opmerkelijk dat het percentage van
positiviteit in ons laboratorium minder werd naarmate meer ervaring werd opgebouwd
met moleculaire testmethodes voor het aantonen van C. pneumoniae. Ook in muticenter
studies werden grote inter- en intra-laboratorium verschillen in de detectie van C.
pneumoniae aangetoond. Deze gezamelijke factoren impliceren dat de resultaten die
verkregen zijn met conventionele PCR methodes, inclusief die van onze studies,
waarschijnlijk beïnvloed zijn door onervarenheid, keuze van DNA polymerase, maar ook
door contaminatie en kruis-reactiviteit.
Terwijl de PCR testen negatief waren voor alle weefsels, detecteerden wij met
behulp van IHC C. pneumoniae MOMP antigenen in de meerderheid van de weefsels
(73,8% in de clarithromycine groep vs. 77,0% in de placebo groep), maar werd
Chlamydia LPS antigeen slechts in één weefsel gevonden. Een overdaad aan MOMP
antigenen in volledige afwezigheid van C. pneumoniae DNA werd ook gevonden door
andere onderzoekers. Het is mogelijk dat C. pneumoniae MOMP antigenen langere tijd
in vaatwandweefsel persisteren, terwijl C. pneumoniae DNA en LPS sneller degraderen.
Een duidelijke aanwijzing vinden wij het feit dat de behandeling van patiënten met
clarithromycine geen invloed had op de MOMP antigeen detectie. Hetzelfde werd
gedemonstreerd in een diermodel experiment, waarbij behandeling met azithromycine
niet geassociëerd was met de eliminatie van Chlamydia antigenen uit vaatwand
monsters van met C. pneumoniae geïnfecteerde konijnen. Ook in een muizenmodel had
121
azithromycine behandeling geen effect op de aanwezigheid van C. pneumoniae
antigenen in de aorta, longen of milt. Met behulp van IHC is het mogelijk om het
microorganisme in specifieke cellen en gebieden te localiseren. De protocollen voor
uitvoering en interpretatie van IHC zijn echter niet gestandaardiseerd. Deze techniek
vereist een subjectieve beoordeling en de interpretatie blijft moeilijk door de
aanwezigheid van achtergrond kleuring en niet-specifieke kleuring.
Het hoge detectie percentage van C. pneumoniae MOMP antigenen en het lage
detectie percentage van C. pneumoniae DNA in vaatwandweefsel zou gedeeltelijk
verklaard kunnen worden door de biologie van de chlamydiae. Infecties met chlamydiae
(bijv. C. trachomatis) worden gekarakteriseerd door afwisselende fases van activiteit en
inactiviteit, gevolgd door fibrosering en verlittekening. In gevorderde infecties zijn
chlamydiae niet levend meer. De lage prevalentie of afwezigheid van C. pneumoniae
DNA en LPS in vaatwand monsters en de aanwezigheid van MOMP antigeen
ondersteunt de theorie dat er geen levende microörganismen aanwezig zijn in
gevorderde atherosclerosis. Volgens deze hypothese zou het eradicatie effect van
antibiotica bestudeerd moeten worden in een beginnend stadium van atherosclerose.
Naast een aangetoond chemotherapeutisch vermogen zouden macrolide
antibiotica een onstekingsremmend effect hebben. Omdat atheromen gekarakteriseerd
worden door laesies die een overdaad aan immuuncellen bevatten, met name
macrophagen en T-cellen, kan atherosclerose gezien worden als een ontstekingsziekte.
Daarnaast zou C. pneumoniae, als levend of dood organisme, een oorzaak kunnen zijn
van
chronische
ontsteking,
waarbij
macrolide
antibiotica
zouden
werken
als
ontstekingsremmende medicatie en zo het cardiovasculaire risico verminderen. Omdat
er veel discussie is over het ontstekingsremmende vermogen van macroliden bij
patiënten met CAV, hebben wij het effect van clarithromycine op onstekingsfactoren
bestudeerd (hoofdstuk 5). De gemiddeld 16 dagen durende behandeling met
clarithromycine had geen invloed op de niveaus van de onstekingsfactoren CRP, IL-2R,
IL-6, IL-8 en TNF-α op de dag van chirurgie en 8 weken later. In onze patiëntengroep
bleek clarithromycine geen ontstekingsremmend effect te hebben. In een interventie
studie waarbij 202 patiënten met instabiele angina of een non-Q-golf myocard infarct
werden behandeld met roxithromycine of placebo, toonden Gurfinkel et al. na 31 dagen
een grotere daling van CRP in de roxithromycine behandelingsgroep in vergelijking met
de placebo groep (p=0,03) aan. Echter waren deze CRP basis niveaus niet
geassocieerd met grote cardiovasculaire incidenten. Anderson et al. vonden een daling
122
SAMENVATTING EN CONCLUSIES
in de combinatie van onstekingsfactoren bij 302 patiënten met antistof titers tegen C.
pneumoniae na azithromycine behandeling. Alhoewel de verschillen in beide studies
statistisch significant zijn, zijn deze verschillen gebaseerd op minimale verandering in de
waarden van de verschillende ontstekingsmediatoren. Een duidelijk ontstekingsremmend effect van macroliden in patiënten met CAV werd niet aangetoond.
In een aantal kleine interventie studies werd aangetoond dat antibiotica
behandeling het cardiovasculaire risico bij patiënten met CAV zou verlagen. Wij volgden
ons cohort patiënten nog gedurende twee jaar na de operatie, om na te gaan of
behandeling met clarithromycine latere cardiovasculaire incidenten en mortaliteit zou
voorkomen. In totaal werden 473 patiënten die op de wachtlijst stonden voor CABG
hartchirurgie uiteindelijk geïncludeerd in de interventie studie, beschreven in hoofdstuk
6. Patiënten werden behandeld voor gemiddeld 16 dagen. Follow-up na 2 jaar werd
volbracht voor 424 van de 473 patiënten. De totale mortaliteit was zo goed als gelijk in
beide groepen (p=0,81). Ook was er geen significant verschil in cardiovasculaire incident
percentages gedurende de follow-up (p=0,86). Behandeling met clarithromycine in
patiënten die wachtten op hartchirurgie, verminderde het aantal daarop volgende
cardiovasculaire events en mortaliteit gedurende 2 jaar follow-up niet. Een review van
alle gerandomiseerd gecontrolleerde trials (RCT’s) tot nu toe in vergelijkbare patiënten
toonde aan dat de meerderheid van de RCT’s geen nuttig effect van een macroliden
kuur in deze patiënten laten zien. De positieve resultaten van kleine trials werden dus
niet bevestigd door grotere studies, inclusief die van ons. Een mogelijke verklaring zou
kunnen zijn dat de kleinere trials mogelijk zijn uitgevoerd in meer geselecteerde
patiëntengroepen wat zou kunnen betekenen dat bepaalde groepen patiënten wel
voordeel hebben van antibiotica. In de WIZARD studie liet analyse in een subpopulatie
een trend zien richting een nuttig effect van antibioticabehandeling bij mannen die roken
of diabetes of hypercholesterolemie hebben. In de subgroep van patiënten die diabetes
hadden en rookten was het percentage cardiovasculaire incidenten 14,6% voor
diegenen die azithromycine kregen, vergeleken met 53% voor diegenen die placebo
kregen. In een subgroep analyse van de ISAR-3 trial hadden patiënten met de hoogste
titers van C. pneumoniae antistoffen een het laagste percentage restenose na
behandeling met roxithromycine in vergelijking met placebo. In de in hoofdstuk 6
beschreven studie bleek behandeling met clarithromycin geen verschillend effect te
hebben bij patiënten met of zonder diabetes, of die wel of niet rookten. Het gebruik van
123
antibiotica als secundaire preventie van atherosclerose kan ons inziens vooralsnog niet
worden aanbevolen.
Het gebruik van antibiotica in het algemeen is geassociëerd met het onstaan van
resistentie tegen deze middelen van vrijwel alle menselijke pathogenen. Resistentie is
een wereldwijd probleem en aanleiding tot grote zorg voor toekomstige therapeutisch
effectiviteit. Daarom is het van belang serieus na te denken bij het gebruik van macrolide
antibiotica, zoals clarithromycine, wanneer deze worden toegepast voor andere doelen
dan de behandeling van infectieziekten. In hoofdstuk 7 wordt het effect van
clarithromycine op de ontwikkeling van resistentie in de oropharyngeale en
nasopharyngeale flora van patiënten die deelnamen aan de interventiestudie.
Resistentieontwikkeling werd gemeten met behulp van agar dilutie (MIC) en door
detectie van resistentie genen (MLS) met PCR. Zestien dagen behandeling met
clarithromycine had een aanzienlijk effect op de ontwikkeling van resistentie in de
oropharyngeale flora en dit effect bleef minstens 8 weken aantoondbaar. Direct na
behandeling was het percentage patiënten met macrolide-resistente flora min of meer
verdubbeld (p<0,0001). Bovendien nam dragerschap van S. aureus significant af. Omdat
de resistentie tegen macroliden reeds aan het toenemen is, hoofdzakelijk door het
toenemend voorschrijven en gebruik van macroliden, zoals clarithromycine, willen wij er
voor pleiten dat macroliden alleen gebruikt worden bij duidelijke indicaties.
Verder bewijs voor de associatie tussen C. pneumoniae en atherosclerose werd
geleverd door diermodel experimenten in een poging de postulaten van Koch te
vervullen. Een probleem is dat een optimaal diermodel niet voor handen is, omdat dieren
van nature geen atherosclerose ontwikkelen. Daarom worden dieren gebruikt die neigen
tot atherosclerose door hyperlipidemie (ApoE muizen, low density lipoprotein muizen of
New Zealand witte konijnen). Introductie van C. pneumoniae in de luchtwegen simuleert
de porte d’ entrée van infectie bij mensen. In deze studies werd aangetoond dat C.
pneumoniae een specifiek tropisme heeft voor de vaatwand, de capaciteit heeft om
ontsteking te induceren en de ontwikkeling van laesies kan initiëren en stimuleren in
hyperlipemische dieren met atherosclerose. Hu et al. namen echter waar dat dit effect
gecorreleerd was met het serum cholesterol niveau van deze dieren. Tevens bleek dat
ernstig hyperlipemische dieren atherosclerose konden ontwikkelen in een bacil-vrije
omgeving. Een causale rol voor C. pneumoniae wordt door deze bevindingen
onwaarschijnlijk. Een bijkomstig tegenargument is dat verschillende andere infectieuze
verwekkers ook de potentie blijken te hebben om vergelijkbare atherosclerotische
124
SAMENVATTING EN CONCLUSIES
veranderingen in hyperlipemische dieren te induceren als C. pneumoniae. Aanvullende
studies hebben het effect van antibioticabehandeling op experimentele atherogenese in
dieren geïnfecteerd met Chlamydia onderzocht. Muhlestein et al. rapporteerde als
eersten dat azithromycine atherosclerose voorkwam in hyperlipemische konijnen die
geïnfecteerd waren met C. pneumoniae. Meer recente observaties laten zien dat
antibioticabehandeling meer effectief is als deze gestart werd kort na experimentele
infectie (< 1 week), wat impliceert dat behandeling met antibiotica geen effect heeft bij
chronische infecties.
Concluderend: aan de hand van de door ons behaalde en in dit proefschrift
beschreven resultaten betwijfelen wij of C. pneumoniae een actieve rol speelt in het
ontstaan van atherosclerose. De pogingen tot het aantonen van de aanwezigheid van C.
pneumoniae in vaatwandmonsters met behulp van verschillende detectiemethoden
gaven controversiële resultaten. Helaas vereisen de beschikbare diagnostische
methodes als PCR en IHC nauwkeurige interpretatie en standaardisatie om een acute,
chronische of persisterende C. pneumoniae infectie aan te tonen of te volgen. Het
gebruik van verschillende detectie technieken leverde geen aanwijzingen voor een
actieve infectie met (levende) C. pneumoniae. De resultaten van onze gerandomiseerd
gecontrolleerde studie ondersteunen het gebruik van antibiotica voor de preventie van
cardiovasculaire events in patiënten met CAV niet. Eradicatie van C. pneumoniae uit
atheroscelrotische laesies kon niet worden aangetoond. Een ontstekingsremmend effect
van de macrolide therapie werd niet waargenomen. Zelfs na 2 jaar was er geen verschil
in het aantal cardiovasculaire incidenten bij patiënten behandeld met clarithromycin of
met placebo. Bovendien veroorzaakte de relatief korte clarithromycinekuur al significante
en blijvende resistentie in de orofaryngeale flora van de behandelde patiënten.
De ware associatie tussen het pathogeen en atherosclerose is nog steeds niet duidelijk,
maar wij achten een oorzakelijke rol van dit microgorganisme erg onwaarchijnlijk in het
kader van het complexe en multifactoriële karakter van atherosclerose.
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PUBLICATIES
PUBLICATIES
PUBLICATIES
1. Kluytmans J, Berg H, Steegh P, Vandenesch F, Etienne J, van Belkum A. Outbreak of
Staphylococcus schleiferi wound infections: strain characterization by randomly amplified
polymorphic DNA analysis, PCR ribotyping, conventional ribotyping, and pulsed-field gel
electrophoresis. J Clin Microbiol 1998;36(8):2214-2219
2. Maraha B, van Der Zee A, Bergmans AM, Pan M, Peeters MF, Berg HF, Scheffer GJ,
Kluytmans JA. Is Mycoplasma pneumoniae associated with vascular disease? J Clin
Microbiol 2000;38(2):935-936
3. Berg HF, Brands WG, van Geldorp TR, Kluytmans-VandenBergh FQ, Kluytmans JA.
Comparison between closed drainage techniques for the treatment of postoperative
mediastinitis. Ann Thorac Surg 2000;70(3):924-929.
4. Maraha B, Berg H, Scheffer GJ, van der Zee A, Bergmans A, Misere J, Kluytmans J, Peeters
M. Correlation between detection methods of Chlamydia pneumoniae in atherosclerotic and
non-atherosclerotic tissues. Diagn Microbiol Infect Dis. 2001;39(3):139-143
5. Berg HF, Van Gendt J, Rimmelzwaan GF, Peeters MF, Van Keulen P. Nosocomial influenza
infection among post-influenza-vaccinated patients with severe pulmonary diseases. Journal
of Infection 2003; 46(2):129-132
6. Berg HF, Maraha B, Bergmans AMC, van der Zee A, Kluytmans JAJW, Peeters MF.
Extraction of Chlamydia pneumoniae-DNA from vascular tissue for use in PCR: an evaluation
of four procedures. Clinical Microbiology and Infection 2003;9(2):135-139
7. Berg HF, Maraha B, Scheffer GJ, Peeters MF, Kluytmans JAJW. The effect of clarithromycin
on inflammatory markers in patients with atherosclerosis. Clinical and Diagnostic Laboratory
Immunology 2003;10(4):525-528
8. Maraha B, Berg HF, Kerver M, Kranendonk S, Hamming J, Kluytmans JAJW, Peeters MF,
van der Zee A. Is the perceived association between Chlamydia pneumoniae and vascular
diseases biased by methodology? Journal of Clinical Microbiology 2004;in press
129
DANKWOORD
DANKWOORD
Het is een mooi moment na al die jaren een proefschrift te kunnen tonen. Dit was
natuurlijk nooit mogelijk geweest zonder de steun van velen om mij heen met wie ik
gedurende het lange traject van het promotieonderzoek heb samengewerkt. Ik wil de
belangrijksten hier bedanken.
Allereerst mijn co-promotor dr. J.A.J.W. Kluytmans. Beste Jan, als gevolg van onze
samenwerking voorafgaand aan mijn co-schappen, was jij degene die me in 1999
opbelde met de vraag of ik voor de Maatschap Artsen-microbioloog Brabant wilde
komen werken om een promotieonderzoek op te starten in de toenmalige Klokkenberg.
Mijn eerste schreden in onderzoek doen en mijn eerste publicatie heb ik aan jou te
danken. Daarna volgde er meer. Het was fijn om met je samen te werken omdat je altijd
wel even tijd had om over het onderzoek mee te denken. Ik heb veel van je geleerd,
door je heldere en kritische blik. Je stimuleerde me ook in mindere tijden door te zetten.
Je overzicht en visie waren erg verhelderend.
Dr. M.F. Peeters. Beste Marcel, als opleider medische microbiologie in Tilburg en
landelijk voorzitter van de Nederlandse Vereniging voor Medische Microbiologie heb je,
materiëel en financiëel, veel ondersteund en voor elkaar gekregen. Je maakte de
uitvoering van diverse studieonderdelen mogelijk. Voor je gedrevenheid, enthousiasme
en onuitputtelijke energie heb ik veel respect. Dankzij jou heb ik de mogelijkheid gehad
de opleiding tot arts-microbioloog te volgen. Ik dank je voor je begrip voor mijn
uiteindelijke heroriëntatie, en de mogelijkheden die je hebt gecreëerd om het onderzoek
uiteindelijk af te ronden.
Mijn promotor Prof.dr. C.M.J.E. Vandenbroucke-Grauls. Beste Christina, de uiteindelijke
samenwerking met jou heeft de voltooiing van dit proefschrift mogelijk gemaakt. Veel
dank voor je heldere kritiek in de laatste fase.
Uit de beginfase op het thoraxcentrum van de Klokkenberg (heden Amphia Ziekenhuis):
W.G.B. Brands. Beste Willem, dankzij onze eerste samenwerking tijdens mijn
afstudeerfase op de Klokkenberg heb ik kennis kunnen maken met de cardiochirurgie en
onderzoek kunnen doen op het gebied van cardiochirurgie en infectieziekten. Je
interesse in mijn reilen en zeilen is altijd een steun geweest.
P.H.J. van Keulen. Beste Peter, dank voor het meedenken en jou ondersteuning bij het
verwerken van de keel- en neuskweken op je bacteriologisch laboratorium in Breda. Alle
kweken zijn keurig ingevroren en konden zo op een later tijdstip verder geanalyseerd
worden.
Prof.dr. G.J. Scheffer. Beste Gert-Jan, tijdens de studieopzet heb je me veel geholpen
met de logistiek op het operatiekamercomplex en lokale laboratorium van de
Klokkenberg en later tevens als mede-auteur.
Anneke Oliemans en Karen van Driel. Jullie huiskamersfeer met immer verse koffie was
heerlijk om af en toe bij te komen in het constante heen en weer gehobbel op de
Klokkenberg. Ik denk nog steeds met veel genoegen aan jullie “moederrol”.
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DANKWOORD
Beste thoraxchirurgen, operatie-assistentes, secretaresses van de polikliniek, analisten
van het laboratorium voor klinisch chemie, arts-assistenten thoraxchirurgie en intensivecare. Veel dank voor jullie medewerking. Met jullie hulp zag ik mijn patiënten wanneer
dat nodig was en werden de juiste materialen en data verzameld.
Uit de daarop volgende fase in het St Elisabeth Ziekenhuis in Tilburg:
Marjolein, Ingrid, Caroline. Jullie taak bij het verwerken van de vele materialen uit deze
studie is onmisbaar geweest. Jullie hebben een hoop werk verzet.
Harold Verbakel, Anneke Bergmans en Anneke van der Zee. Het regelmatige overleg
met jullie en jullie kennis van de moleculaire microbiologie tijdens de constante
progressie van het onderzoek is zeer nuttig en vernieuwend geweest.
De analisten van het bacteriologisch laboratorium. Ik dank jullie voor jullie hulp bij mijn
haast eindeloze aantallen kweken.
Dr. P.J. Kabel. Beste Peter, dank voor het uitzoeken en realiseren van de cytokine
bepalingen. Er is uiteindelijk een hoofstuk uit ontstaan.
De Maatschap Artsen-microbioloog Brabant. Dank voor jullie begrip en de mogelijkheid
om naast de opleiding aan het onderzoek door te werken.
Secretaresses van de Medische Microbiologie. Dank voor het faciliteren in de diverse
secretariële taken die het onderzoek met zich meebrengt.
Beste Boulos Maraha. Van het begin tot het eind heb ik met je samengewerkt.
Aanvankelijk, jij op het laboratorium en ik in de kliniek. Het is mooi dat het ons
uiteindelijk gelukt is twee proefschriften te schrijven. Het is bijzonder beide vader te
worden in dezelfde periode en op dezelfde datum het proefschrift te verdedigen. Dank
voor de vruchtbare samenwerking.
Beste Annemarie en Bram. In de laatste fase van het schrijven heb ik veel steun, begrip
en lol met jullie gehad. Leuk dat ik in mijn “niche” kon blijven werken. En maar schaapjes
tellen.
De eindfase in de huisartsenpraktijk:
Beste Jan Corten en Bart van West. Jullie flexibiliteit en begrip voor mijn dubbele
agenda naast de werkzaamheden in de praktijk waren onmisbaar bij de afronding van
mijn proefschrift. Heel veel dank daarvoor.
De rode draad:
Lieve Noortje, je hebt veel geduld en begrip voor me gehad de afgelopen tijd. Jouw
heldere kijk op een tekst is erg nuttig gebleken, ook al spreekt het onderwerp niet altijd
tot de verbeelding. Je bent de onmisbare factor in dit geheel geweest.
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CURRICULUM VITAE
CURRICULUM VITAE
Hans F. Berg is geboren op 19 september 1969 te Breda. Na het VWO op de Nassau
Scholengemeenschap in Breda begint hij in 1990 met de studie geneeskunde aan de
Rijks Universiteit Leiden. Tijdens zijn studie werkt hij als student assistent (Joshua) op
de Intensive Care Unit van de afdeling thoraxchirurgie van het Leids Universitair
Medisch Centrum, waar hij patienten verzorgt op hun eerste postoperatieve dag na
openhart chirurgie (hoofd: prof. dr. H. A. Huysmans). In 1996 begint hij een
afstudeeronderzoek naar postoperatieve wondinfecties na openhart chirurgie op de
afdeling thoraxchirurgie van het Medisch Centrum de Klokkenberg (momenteel
onderdeel van het Amphia Ziekenhuis) in Breda. Daarbij werkt hij nauw samen met de
afdeling medische microbiologie (W. Brands en dr. J.A.J.W. Kluytmans). In 1997 behaalt
hij zijn doctoraalexamen. Na zijn co-assistentschappen volgt begin 1999 het artsexamen. In datzelfde jaar begint hij als arts-onderzoeker op de Klokkenberg te Breda en
met de studies beschreven in dit proefschrift. Twee jaar later start hij de opleiding
medische microbiologie in het St Elisabeth Ziekenhuis te Tilburg (opleider: dr. M. F.
Peeters). In 2003 verandert zijn beroepskeuze en begint hij met de opleiding
huisartsgeneeskunde aan het Erasmus Medisch Centrum in Rotterdam. Tegelijkertijd
schrijft hij dit proefschrift.
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