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Failure to detect Chlamydia pneumoniae DNA in cerebral aneursymal sac tissue with two different polymerase chain reaction methods
  1. S Cagli1,
  2. N Oktar1,
  3. T Dalbasti1,
  4. S Erensoy2,
  5. N Özdamar1,
  6. S Göksel2,
  7. A Sayiner3,
  8. A Bilgiç2
  1. 1Department of Neurosurgery, Ege University School of Medicine, Izmir, Turkey
  2. 2Department of Clinical Microbiology, Ege University School of Medicine
  3. 3Dokuz Eylül University, Izmir, Turkey
  1. Correspondence to:
 Professor N Oktar, Ege Üniversitesi Tip Fak Hast, Nörosirürji AbD, Bornova, lzmir TR35100, Turkey; 
 noktar{at}med.ege.edu.tr

Abstract

Objective:Chlamydia pneumoniae (C pneumoniae) is a common cause of a usually mild, community acquired pneumonia. This organism, however, can spread from the respiratory tract into other parts of the body and has been detected in up to 70% of atheromatous lesions in blood vessels. Although the exact mechanism of the C Pneumoniae contribution to the pathogenesis of atherosclerosis remains unknown, prophylactic antibiotic trials are planned for people at high risk for coronary disease.

Method: In this study the authors aimed to investigate C pneumoniae DNA content in the cerebral aneurysmal sac tissue with the aid of polymerase chain reaction (PCR) method. C pneumoniae DNA was searched in 15 surgically clipped and removed aneurysmal sac tissue and in two tumour (an ependymoma of the fourth ventricle and a craniofaringoma) samples by touchdown enzyme time release PCR (TETR PCR) targeting 16S rRNA gene and by nested PCR targeting ompA gene.

Results: Both PCR methods were sensitive to detect in C pneumoniae 4×10−2 genomes. C pneumoniae DNA was not detected in any of the 17 sample tissues of these patients.

Conclusion: The contribution of C pneumoniae in the development of intracranial aneurysms cannot be excluded despite the results of this study. Further studies on the possible role of C pneumoniae or any other micro-organisms in the pathogenesis of aneurysms should be performed.

  • cerebral aneurysm
  • polymerase chain reaction
  • chlamydia pneumoniae
  • PCR, polymerase chain reaction
  • TETR PCR, touchdown enzyme time release polymerase chain reaction

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Chlamydia pneumoniae is a common cause of a usually mild, community acquired pneumonia. This organism, however, can spread from the respiratory tract into other parts of the body and has been detected in up to 70% of atheromatous lesions in blood vessels.1 Although the exact mechanism of the contribution of C pneumoniae to the pathogenesis of atherosclerosis remains unknown, prophylactic antibiotic trials are planned for people at high risk for coronary disease2 and even for the abdominal aortic aneurysm.3 For years chlamydia were thought to lack key enzymes and cellular machinery for generating ATP, instead sequestering host nucleoside triphosphates by translocation mechanism. The new sequence does reveal two potential ATP/ADP translocases, but it also identifies genes that may allow chlamydia to generate at least minimal amounts of ATP on its own. A unique family of 18 surface exposed outer membrane proteins (POMPs) were recently identified in the unfinished C genome.4

In recent studies investigating the presence of C pneumoniae DNA in the wall of abdominal aortic aneurysms (AAA) authors5–8 concluded that C pneumoniae was often present in AAA in a viable form and that C pneumoniae was linked to the pathogenesis of AAA. On the other hand some studies failed to detect C pneumoniae in symptomatic AAA9 or any clinical correlation between atherosclerotic plaque behaviour in patients with established carotid artery stenosis (tables 1 and 2).10

Table 1

C pneumoniae in abdominal aorta aneurysms (AAA)

Table 2

C pneumoniae in arteriectomy material

In this study we aimed to determine and analyse C pneumoniae DNA content in the cerebral aneurysmal sac tissue with the aid of polymerase chain reaction (PCR) method.

METHODS

The study group consisted of 15 patients operated transcranially for cerebral aneurysm (six women, nine men, mean age 48.7 years) (table 3). Specimens from the aneurysm wall were taken peroperatively, after prompt clipping under sterile conditions. Two tumour specimens (an ependymoma of the fourth ventricle and a craniopharingioma) were also collected for C pneumoniae DNA analyses (table 4). The specimens from these groups were frozen at −70°C immediately after collection.

Table 3

Aneurysm cases

Table 4

Tumour cases

DNA extraction

C pneumoniae DNA from tissue samples was extracted with High Pure PCR Template Preperation Kit (Roche Diagnostics, Indianapolis, USA).

PCR

Touchdown enzyme time release (TETR) PCR targeting the 16S rRNA gene and a nested PCR targeting the ompA gene were performed to detect C pneumoniae DNA. All amplification reactions were done in a volume of 50 μl containing 200 μM of four deoxynucleoside triphosphates, 1.5U of AmpliTaq Gold DNA polymerase (Amplitaq Gold; Perkin-Elmer, Branchburg, NJ, USA). Different concentrations of primers and MgCl2 were used for each PCR. Amplification reactions were performed on a GeneAmp9600 thermocycler (Perkin-Elmer, Branchburg, NJ, USA) using different cycling conditions for each PCR. Published PCR protocols were used for both TETR PCR17 and nested PCR18 with some modifications. PCR primers tested were CPN90 5′ GGT CTC AAC CCC ATC CGT GTC GG 3′, CPN91 5′ TGC GGA AAG CTG TAT TTC TAC AGT T 3′, CP1 5′ TTA CAA GCC TTG CCT GTA GG 3′, CP2 5′ GCG ATC CCA AAT GTT TAA GGC 3′, CPC 5′ TTA TTA ATT GAT GGT ACA ATA 3′ and CPD 5′ ATC TAC GGC AGT AGT ATA GTT 3′.

Briefly TETR PCR was performed using CPN90-CPN91 primer pair with a 0.25 μM concentration of each primer, 2.5 mM MgCl2 and 20 μl of the extracted DNA. Cycling protocol was 75 seconds at 95°C, followed by 60 cycles of denaturation at 94°C for 45 seconds, annealing beginning at 64°C and ending at 52°C for 45 seconds, and extension at 72°C for one minute. The annealing temperature was lowered 10°C every four cycles until 52°C and this temperature was kept until the end of the cycling process.

CP1-CP2 primers with nested pair CPC-CPD were used for the ompA nested PCR. The first round of amplification used 1.5 mM MgCl2, 0.4 μM of each primer and 20 μl of the extracted DNA. Cycling consisted of nine minutes at 95°C for Taq polymerase activation, 20 cycles of one minute at 94°C, one minutes at 65°C (temperature was decreased 0.5°C for each cycle) and one minute at 72°C plus an additional 20 cycles of one minute at 94°C, one minute at 55°C and one minute at 72°C. The PCR products amplified by the outer primer pair were diluted 1:5 and 5 μl was added to a new PCR mixture containing 1 μM of each primer and 3 mM of MgCl2.

Cycling protocol entailed nine minutes at 95°C for Taq polymerase activation, 30 cycles of one minute at 94°C, one minute at 50°C and one minute at 72°C.

Detection of the human β globin gene

For extraction and PCR inhibition control, a fragment of the human β globin gene was amplified as previously described.19

All amplification products were analysed by agarose gel electrophoresis and ethidium bromide staining. The expected amplicon sizes were 197bp for TETR PCR, 333bp for ompA outer primer pair, 207bp for ompA inner primer pair and 536 bp for the human β globin gene.

RESULTS

Two different PCR assays were performed to detect C pneumoniae DNA. Both TETR PCR and ompA nested PCR was sensitive to detect 10−3 colony forming units. All samples were negative for C pneumoniae DNA (fig 1 and 2). A 536bp fragment of the human β globin gene was detected in all of the samples except one (fig 3).

Figure 1

C pneumoniae TETR PCR of clinical samples. Lanes 1 to 3, 5 to 7 clinical samples. Lanes 4 and 8 negative control (water). Lanes 9 and 11 positive control (C pneumoniae 4×10−1 and 4×10−2 CFU). Lane 10 water. Lane 12 DNA molecular weight marker (XIV; 100 bp ladder, Roche Diagnostics).

Figure 2

C pneumoniae OmpA PCR of clinical samples. Lanes 1 to 7 clinical samples. Lane 8 negative control (water). Lanes 9 to 11 positive controls (C pneumoniae 40, 4, and 4×10−1 CFU respectively). Lane 12 DNA molecular weight marker (XIII; 50 bp ladder, Roche Diagnostics).

Figure 3

β globin control PCR. Lane 1 to 5 and 7 to 11 clinical samples. Lane 6 negative control (water). Lane 12 DNA molecular weight marker (XIV; 100 bp ladder, Roche Diagnostics).

DISCUSSION

The term “mycotic aneurysm” was first used by Osler in 1885.20 Subsequently Stengel and Wolferth (1923) reported 217 cases, including their own, of aneurysms developing during bacterial infections.21 Among these 42 cases had intracranial aneuryms, and since that time at least 30 cases of mycotic aneurysms have been reported.22 A review of 1126 cases of intracranial aneuryms in 193923 showed an incidence of 6% mycotic aneurysms. After the introduction of antibiotic treatment of subacute bacterial endocarditis, bacterial cerebral aneurysms have become quite rare. As Yasargil stated “...A more likely error in present-day diagnosis is the failure to consider an aneurysm to be of bacterial embolic origin...”.24 Before the antibiotic era, cerebral aneurysms secondary to infected emboli from the vegetations of endocarditis were not uncommon.

At the beginning of the past century, mycotic aneurysms were thought to account for about one quarter of intracranial aneurysms.24,25

The term “arteriosclerotic aneurysm” is used to describe fusiform dilatation of a cerebral vessel in which the wall has undergone atheromatous degeneration. The first comprehensive discussion of this entity was by Dandy in 1944, who encountered 11 cases of elongated and tortuos vascular arteries in the course of posterior fossa procedures.26 Such serpentine dilatation of the vertebral basilar or internal carotid arteries is a frequent occurence in patients with severe atherosclerosis.27 These lesions are usually classified as atherosclerotic aneurysms, although they are not necessarily associated with senile ectasia or atherosclerosis of the vessel, for example, Marfan syndrome, megalodolichobasilaris or idiopathic mediannecrosis. Atherosclerotic aneurysms accounted for about 50% of the lesions in older statistics28 and are found in 8% to 16% of Housepian and Pool (1958) and Jellinger’s (1979) series.27,29 Ohara et al (1979) distinguished two types of arteriosclerotic aneurysms.30 One is the type where the trunk arteries, such as the basilar artery, themselves, swell to a fusiform shape (Fusiform aneurysm). The other is the type where a saccular aneurysm arises having no relation to the arterial forks with considerable sclerosis of the parental artery. In Suzuki’s series (1979) eleven of 1116 aneurysm cases (1%) had sclerotic aneurysms.31

C pneumoniae has been associated with atherosclerotic cardiovascular disease by both seroepidemiological studies and direct detection of the organism in atherosclerotic plaque by electron microscopy, immunocytochemistry, and PCR.32 The role of inflammatory reactions in the pathogenesis of atherosclerosis is widely accepted. Recently, an increasing body of evidence has linked infections to atherosclerosis. It is hypothesised that infections could interact with other risk factors of vascular disease, increasing the endothelial damage and the production of atherosclerotic plaques. Several different infectious agents have been related to the atherosclerosis genesis: mainly herpesvirus, Helicobacter pylori, and C pneumoniae. Several lines of evidence strongly link C pneumoniae to atherosclerosis. Consequently, several studies evaluating the effectiveness of antibiotic treatment in the reduction of cardiac ischaemic events in patients with C pneumoniae seropositivity have been performed.

On the other hand several reports show lack of association between seropositivity to C pneumoniae and carotid atherosclerosis33 and even C pneumoniae antibodies and high lipoprotein α levels do not predict ischaemic cerebral infarctions.34 In contrast with previous published papers, Nobel et al could not confirm an association of C Pneumoniae infection with an acute coronary event.35 Gibbs et al research showed that the presence of the infectious organism has little detectable impact on plaque instability when measured by clinically significant markers.10 This raises important questions for the rationale of antibiotic therapy in atherosclerosis.36 The validity of the hypothesis that infection contributes to atherosclerosis has not been definitively established, although the evidence is becoming compelling, with several interesting studies presented at the 2001 ACC meeting (S E Epstein, 50th annual scientific session of the American College of Cardiology, 19 March 2001). Evidence is also acumulating that autoimmune responses, perhaps triggered by infection, may be one of the mechanisms contributing to atherogenesis. The concepts are intriguing and will undoubtedly serve as the focus of many investigative studies presented at future meetings (50th annual scientific session of the American College of Cardiology). In the study of Vink et al the prevalences of C pneumoniae at multiple locations in the arterial system within the same person were observed as highest in the abdominal aorta (67%), internal and common iliac arteries (41%), and coronary arteries (33%). The lowest prevalences were observed in the radial (0%) and cerebral (2%) arteries.16 C pneumoniae was mostly observed at locations that are related to clinically relevant features. Within the patient group, the distribution of C pneumoniae is associated with the distribution of atherosclerosis. The role of the micro-organism in atherosclerotic disease remains to be elucidated. This study and our results with cerebral aneurysms show at least the possible role of C pneumoniae in the patogenesis of the cerebral aneurysms.

In this study, negative results of PCR may be discussed (or questioned) for the presence of PCR inhibitors in the samples or the inefficiency of the PCR. For the assesment of these factors; fragment of human β globin gene was investigated in every sample as an endogenous control. The sensitivity of the PCR methods was determined with positive controls of C pneumoniae culture samples with a known genomes. All the samples were found to be positive for β globin gene indicating the absence of PCR inhibitors. Performed TETR PCR and ompA PCR methods were found to be as sensitive to detect 4×10−2 genomes per reaction. We cannot exclude the contribution of C pneumoniae in development of intracranial aneurysms despite the result of this study. Further studies on the possible role of C pneumoniae or any other micro-organisms (such as H pylori, citomegalovirus, autoantibodies against “heat shock” proteins, hepatitis A virus, herpes simplex virus 1 and 2 (HSV1–2), Porphyromonas gingivalis, et al) in the pathogenesis of aneurysms should be performed.

REFERENCES

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Footnotes

  • Competing interests: none declared.

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