European Journal of Clinical Microbiology & Infectious Diseases

, Volume 26, Issue 2, pp 141–145

Lack of microbial DNA in tissue specimens of patients with abdominal aortic aneurysms and positive Chlamydiales serology

Authors

  • B. Falkensammer
    • Department of Hygiene, Microbiology and Social MedicineInnsbruck Medical University
  • C. Duftner
    • Department of Internal MedicineGeneral Hospital of the Elisabethinen Klagenfurt
  • R. Seiler
    • Department of Surgery, Division of Vascular SurgeryInnsbruck Medical University
  • M. Pavlic
    • Institute of Legal MedicineInnsbruck Medical University
  • G. Walder
    • Department of Hygiene, Microbiology and Social MedicineInnsbruck Medical University
  • D. Wilflingseder
    • Department of Hygiene, Microbiology and Social MedicineInnsbruck Medical University
  • H. Stoiber
    • Department of Hygiene, Microbiology and Social MedicineInnsbruck Medical University
  • P. Klein-Weigel
    • Department of Surgery, Division of Vascular SurgeryInnsbruck Medical University
  • M. Dierich
    • Department of Hygiene, Microbiology and Social MedicineInnsbruck Medical University
  • G. Fraedrich
    • Department of Surgery, Division of Vascular SurgeryInnsbruck Medical University
    • Department of Hygiene, Microbiology and Social MedicineInnsbruck Medical University
  • M. Schirmer
    • Department of Internal MedicineGeneral Hospital of the Elisabethinen Klagenfurt
  • on behalf of the Innsbruck Abdominal Aortic Aneurysm Trial-Group
    • Department of Internal MedicineInnsbruck Medical University
Concise Article

DOI: 10.1007/s10096-006-0245-5

Cite this article as:
Falkensammer, B., Duftner, C., Seiler, R. et al. Eur J Clin Microbiol Infect Dis (2007) 26: 141. doi:10.1007/s10096-006-0245-5

Abstract

In a case-control study that included a total of 98 patients and 83 controls, the possible link between various pathogens and abdominal aortic aneurysms was investigated. For 68 patients with abdominal aortic aneurysm and age-matched controls, no differences were detected in the levels of immunoglobulin (Ig)A and IgG Chlamydiaceae and Chlamydophila pneumoniae antibodies. Patients with IgA titers positive for Chlamydophila pneumoniae showed progressive disease (defined as an annual increase of the aneurysm diameter of ≥0.5 cm) more frequently than patients with negative IgA titers (p = 0.046). Polymerase chain reactions performed to detect DNA for Chlamydophila pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, human cytomegalovirus, Borrelia burgdorferi and Helicobacter pylori in tissue specimens of 30 patients and 15 controls were negative. In summary, Chlamydophila pneumoniae may contribute to aortic aneurysm disease progression, but DNA of this and other pathogens was not found in patients’ specimens.

Introduction

The pathogenesis of abdominal aortic aneurysms (AAAs) has not been completely clarified to date [1]. Several studies have examined microorganisms including Chlamydophila (Cp.) pneumoniae, Helicobacter (H.) pylori and human cytomegalovirus (HCMV) as possible triggers for the development of AAAs, but the data are still inconclusive [2]. According to the current literature Cp. pneumoniae appears to be the microorganism most likely to be involved in the pathogenesis of AAAs. Cp. pneumoniae is not only an important human pathogen causing acute respiratory diseases; numerous publications report a link between Cp. pneumoniae infection and atherosclerosis [3, 4], which is a major cause of cardiovascular disease and death in industrialized countries. However, other authors could not verify that this pathogen was the cause of arterial calcification or sclerosis [5, 6]. In addition to infecting the epithelial cells of the human respiratory tract, Cp. pneumoniae may also infect macrophages, vascular endothelial cells and smooth muscle cells, which are the key players in human atherosclerosis [7]. Cp. pneumoniae can also increase cholesterol accumulation in infected macrophages [8], but it is still unknown whether a causal relationship between chlamydial infections and atheromatous changes exists. Other pathogenic agents, including HCMV and H. pylori, have also been discussed as potentially causative in atherosclerotic development [3, 4]. The aim of this study was to investigate the presence of infectious pathogens in AAA tissue samples compared to atherosclerotic and normal aortic specimens and to discuss the results in light of serological data.

Patients and methods

In this case-control study, 68 consecutive patients with a sonographically determined AAA (diameter >3 cm) were recruited from our vascular surgery outpatient clinic. Patients with any history of an autoimmune disease or mycotic AAA were excluded. This study was part of a cooperative program of the Innsbruck Abdominal Aortic Aneurysm Trial-Group and was approved by the local Ethics Committee of the Innsbruck Medical University. All patients gave informed and written consent prior to enrollment into the study.

Chlamydial serology was analyzed in the peripheral blood of 68 AAA patients (age 74.4 ± 8.2 years) enrolled at the Division of Vascular Surgery and of 68 healthy age-matched controls (age 73.7 ± 7.8 years). In the group of AAA patients the mean erythrocyte sedimentation rate was 19.3 ± 22.4 mm/h (normal range <15), the mean C-reactive protein level was 2.6 ± 5.1 mg/dl (normal range <0.7) and the mean leukocyte count was 7.8 ± 3.1 G/l (normal range 3.8–10.5). Disease progression was defined as an annual increase in the AAA diameter of ≥0.5 cm and was routinely measured by sonography.

The enzyme-linked immunosorbent assay technique was used for quantitative determination of specific IgG and IgA antibody levels against chlamydial lipopolysaccharides (Chlamydiaceae; Chl. spp) (Medac, Hamburg, Germany) and Cp. pneumoniae IgG and IgA (Ani Labsystems, Oy, Helsinki, Finland) in human sera. The latter specific assay detects antibodies directed against the major outer membrane protein (momp) of Cp. pneumoniae of types IgG and IgA, depending on the conjugate used. All tests were performed according to the manufacturers’ instructions and the cutoff values were calculated as recommended by the manufacturers. The results were expressed as enzyme immunounits (EIU) using the following formula for calculations (Abs = Absorbance):
$$ {\text{EIU}} = {{\left( {{\text{Abs}}_{{{\text{sample}}}} - {\text{Abs}}_{{{\text{blank}}}} } \right)}} \mathord{\left/ {\vphantom {{{\left( {{\text{Absorbance}}_{{{\text{sample}}}} - {\text{Absorbance}}_{{{\text{blank}}}} } \right)}} {{\left( {{\text{Absorbance}}_{{{\text{calibrator}}}} - {\text{Absorbance}}_{{{\text{blank}}}} } \right)}}}} \right. \kern-\nulldelimiterspace} {{\left( {{\text{Abs}}_{{{\text{calibrator}}}} - {\text{Abs}}_{{{\text{blank}}}} } \right)}} \times 30 $$

AAA specimens with an increased maximal diameter of >5 cm were also obtained during surgery from 30 additional patients; however, these patients were not included in the serological analysis of the study. Among these 30 patients, 10.7% were symptomatic for AAA and 7.1% of the AAAs were ruptured or perforated. Control abdominal aortic tissues were obtained post mortem from 15 individuals without AAA disease. Six control specimens showed macroscopic sclerotic changes, whereas in nine other control specimens no macroscopic atheromatous plaques were visible or palpable in the aortic wall. DNA was extracted separately from 25 mg of tissue specimen from AAA patients and controls using the DNeasy Tissue Kit (Qiagen, Hilden, Germany).

For real-time PCR testing, all tissue samples were tested for the quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA as a “housekeeping” control gene [9] using the iCycler (Bio-Rad, Hercules, CA, USA) with the corresponding primers and probes (Table 1) and the Brilliant® QPCR Core Reagent Kit (Stratagene, La Jolla, CA, USA). All samples were then tested twice for the presence of chlamydial-specific DNA. BrilliantSYBR® Green QPCR Master Mix (Stratagene) was mixed with specific primers (Table 1) for a nested PCR targeting the outer membrane protein (omp) A gene and a conservative part of the 16S-rRNA gene [10]. Sixty randomly assigned extracts of aortic-tissue DNA from two opposing, independent sites of the AAAs of 30 patients were tested for DNA from Chlamydia (C.) trachomatis, Chlamydophila (Cp.) psittaci, H. pylori, Borrelia (B.) burgdorferi and HCMV. DNA extracts were amplified with appropriate primers and probes listed in Table 1 as well as either SYBR® Green QPCR Master Mix or Brilliant® QPCR Core Reagent Kit. For every PCR positive as well as no-template controls were used.
Table 1

Primer sequences used to test tissue samples

Primer

Primer sequence

C (nmol/l)

Genome region

Chlam for

5′–CTG AAA CCA RTA GCT TAY AAG CGG T–3′

200

16S-rRNA

Chlam rev

5′–AYC TCG CCG TTT ARC TTA ACT CC–3′

200

16S-rRNA

Chlam probe

5′–Fa–CTC ATC ATG CAA AAG GCA CGC CG–Tr–3′

125

16S-rRNA

Pneum for1

5′–GCI YTI TGG GAR TGY GGI TGY GCI AC–3′

400

ompA

Pneum rev1

5′–TTA GAA ICK GAA TTG IGC RTT IAY GTG IGC IGC–3′

400

ompA

Pneum for2

5′–GGI GCW GMI TTC CAA TAY GCI CAR TC–3′

400

ompA

Pneum rev2

5′–GTA CTC CAA TGT ATG GCA CTA AAG A–3′

400

ompA

Trach for

5′–GGI GCW GMI TTC CAA TAY GCI CAR TC–3′

400

ompA

Trach rev

5′–ACC ATT TAA CTC CAA TGT ARG GAG TG–3′

400

ompA

Psitt for

5′–GTA ATT TCI AGC CCA GCA CAA TTY GTG–3′

400

ompA

Psitt rev

5′–CCR CAA GMT TTT CTR GAY TTC AWY TTG TTR AT–3′

400

ompA

Borr for

5′–GAA TTA GCA GTT CAA TCA GG–3′

750

Flagellin gene

Borr rev

5′–TTC GTC TGT AAG TTG CTC TAT–3′

750

Flagellin gene

Borr probe

5′–Fa–AAC GGC ACA TAT TCA GAT GCA GAC A–Tr–3′

150

Flagellin gene

H. pylori for

5′–TTC AAA TCG GCT CAC ACT TC–3′

250

Urease A gene

H. pylori rev

5′–CTG TTA CCG CCA ATG TCA AT–3′

250

Urease A gene

HCMV for

5′–GGC AAC TCG GCC TAC GAG T–3′

200

Glycoprotein B

HCMV rev

5′–TGG AGA TGC TGC TGA GGT CA–3′

200

Glycoprotein B

HCMV probe

5′–Fa–CGT GGA CTA CCT CTT CAA ACG CAT G–Tr–3′

125

Glycoprotein B

GAPDH for

5′–CTC ATG ACC ACA GTC CAT GC–3′

200

Human GAPDH

GAPDH rev

5′–CAC GCC ACA GTT TCC CG–3′

200

Human GAPDH

GAPDH probe

5′–Fa–CAG AAG ACT GTG GAT GGC CCC–Tr–3′

125

Human GAPDH

for forward, rev reverse, C primer concentration, Fa 6-carboxy-fluorescein, GAPDH glyceraldehyde-3-phosphate dehydrogenase, Tr 6-carboxy-teremethyl-rhodamine

Data are expressed as mean±SD for continuous variables. The Mann-Whitney and the Fisher’s exact test were used as appropriate. Statistical analyses were performed using SPSS for Windows version 11.0 (SPSS, Birmingham, UK). A p value of <0.05 was considered statistically significant. All reported p values are two-sided.

Results and discussion

Cases and controls were tested for IgG and IgA antibodies against Chl. spp. and Cp. pneumoniae. The IgG antibody levels of the AAA patients were no different than those of the controls. IgG levels for Chl. spp were 175.7 ± 163.7 EIU in the AAA group and 153.3 ± 158.6 EIU in the control group. Using an IgG cut-off level of <120 EIU for Chl. spp., 47.1% of AAA patients were positive compared with 36.8% of the controls. IgG levels for Cp. pneumoniae were 121.6 ± 107.1 EIU in the AAA group compared to 115.7 ± 82.2 EIU in the control group. Using an IgG cut-off level of <45 EIU for Cp. pneumoniae, 75.0% of AAA patients were positive compared with 83.8% of controls.

IgA antibody levels against Chl. spp were 79.5 ± 66.7 EIU in the AAA group and 73.0 ± 75.8 EIU in the control group. Using an IgA cut-off level of <60 EIU for Chl. spp, 44.1% of AAA patients were positive compared with 36.8% of controls. Against Cp. pneumoniae the IgA antibody levels were 25.0 ± 22.5 EIU in AAA patients and 28.5 ± 24.7 EIU in the controls. Using an IgA cutoff level of <12 EIU for Cp. pneumoniae, 69.1% of AAA patients were positive compared with 75.0% of controls.

Follow-up data were available for 47 patients with AAA disease. Between months 3 and 42 of follow-up (18.8 ± 11.1 months), 32 AAA patients showed progressive disease, as defined by an annual increase of the AAA diameter of ≥0.5 cm/year, whereas 15 patients were considered as having stable disease. AAA patients with positive IgA antibody titers against Cp. pneumoniae were more likely to have progressive disease (25/32, 78.1%) than patients with negative IgA titers (7/15 patients, 46.7%, p = 0.046) (Fig. 1). No correlations were found between disease progression and either positive IgG titers against Cp. pneumoniae or IgA and IgG titers against Chl. spp.
https://static-content.springer.com/image/art%3A10.1007%2Fs10096-006-0245-5/MediaObjects/10096_2006_245_Fig1_HTML.gif
Fig. 1

Chlamydiaceae spp and Cp. pneumoniae serology and AAA disease progression. Fisher’s exact test showed disease progression more frequently in AAA patients with positive Cp. pneumoniae IgA antibody titers [IgA positive] (78.1%) than in AAA patients with negative IgA titers [IgA negative] (46.7%, p = 0.046)

In all 90 tissue extracts, similar amounts of GAPDH DNA were detected. The mean threshold cycle (Ct) values were 22.5 ± 4.7; however, no specific DNA was amplified for Cp. pneumoniae, Cp. trachomatis, C. psittaci, H. pylori, B. burgdorferi or HCMV in the tested pathologic samples or in the control aortic specimens. Specific DNA, however, was detectable in all positive controls. Melting curve analysis of SYBR®Green PCR amplification of positive controls showed melting temperatures of 84°C for Cp. pneumoniae, 82°C for C. trachomatis, 81°C for Cp. psittaci and 82°C for H. pylori.

In this case-control study, patients with progressive AAA disease showed pathologic levels of Cp. pneumoniae IgA antibodies more often than AAA patients with stable disease (Fig. 1). These findings confirm results of earlier studies [11, 12]. No significant correlation was found between Cp. spp antibodies and disease progression.

Using real-time PCR, Chlamydiales-specific DNA was not detected in any of the opposing sites of the AAA specimens. These results support the PCR findings of Lindholt et al. [2]. Other PCR studies found 51–100% of tissue specimens positive for Cp. pneumoniae using PCR [2]. The location of the primer pairs in the 16S-rRNA region or in a region coding for an outer membrane protein (omp, momp) does not appear to account for the differing results. At present we can only speculate about the underlying reasons for these differences. All of our real-time PCR runs were valid with detectable DNA in positive controls, correct melting temperatures and no DNA detection in no-template controls. The lack of specific DNA for chlamydial spp and C. trachomatis, Cp. psittaci, H. pylori, B. burgdorferi and HCMV in AAA tissue samples reduces the probability of a local pathogenetic role of these microorganisms in the development of AAAs.

The association of positive IgA antibody titers against Cp. pneumoniae and AAA disease progression on the one hand but lack of specific chlamydial DNA on the other hand, points towards a systemic rather than a local role of Cp. pneumoniae in AAA disease progression. Whether disease activity is triggered by lipopolysaccharides from gram-negative bacteria via the stimulation of Toll-like receptors, as observed in giant cells arteritis [13], or the described discrepancy relies on other mechanisms, such as molecular mimicry, remains elusive so far. The finding of antibodies against Cp. pneumoniae outer membrane protein in serum cross-reacting with the heavy chain of immunoglobulin in AAA patients further supports the hypothesis of an underlying autoimmune process, perhaps triggered by an initial Cp. pneumoniae infection [14].

This study was limited by incomplete follow-up data for the AAA patients, which affected the power of the calculated statistics and indicates that different commercially available serological tests may not necessarily produce congruent results [15].

In conclusion, infections with Cp. pneumoniae may contribute to the progression of AAA disease. Nevertheless, in this study DNA of Cp. pneumoniae, C. trachomatis, Cp. psittaci and H. pylori, B. burgdorferi and HCMV was not detected in any of the specimens obtained from patients with AAA.

Acknowledgments

The study was supported by the European Union (QLK-CT-2002-00882 and LSHB-CT-2005-512061), the Research Fund of the Austrian National Bank (no. 8825, no. 9715), the Tyrolean Research Fund (project no. 0404/121), the Ludwig Boltzmann Society, the Federal State of Tyrol, and the “Verein zur Förderung der Hämatologie, Onkologie und Immunologie,” Innsbruck, Austria. We wish to thank the technical assistants from the virological–serological laboratory for their helpful support.

Copyright information

© Springer-Verlag 2007