Molecular microbiological findings in aneurysm walls

Using RT-qPCR, bacteria were detected in 49/70 (70%) of the aneurysms (Table 3).

The prevalence of bacteria was 69% in the ruptured aneurysm wall samples and 71%

in the unruptured aneurysm wall samples. In the ruptured group, endodontic and periodontal pathogens were identified in 25/42 (60%) and 18/42 (43%) of aneurysms, respectively. Figure 2 shows the frequencies of bacterial DNA-positive findings in all cases (42 ruptured and 28 unruptured samples) from Studies I and II.

In ruptured aneurysm wall samples, 13 out of 42 (31%) contained the DNA of both periodontal and endodontic bacteria. In the unruptured aneurysm wall samples, 43%

contained DNA from both types of bacteria. Patients with an unruptured aneurysm but with a history of previous SAH had bacterial findings similar to those of the patients without a history of SAH (total positive 71% and 83%, respectively; p=0.62;

Fisher´s test).

Bacterial DNA from the Streptococcus mitis -group and Fusobacterium nucleatum were the most common pathogens. Two separate n-fold values for the amount of bacterial DNA were calculated in each aneurysm sample; the first used the mean amount of bacterial DNA from the LITA samples as a control, and the second used the individual’s own blood sample as a control. Figure 3 shows the strong correlation between these two values (r=0.84; p<0.001). The total amount of bacterial DNA in the aneurysm tissue samples was 12.2-times higher than that found in their control blood samples (mean 12.2; sd 25.9; median 4.1). The amount of bacterial DNA was 9.1 times higher in the aneurysm tissue samples than that found in the LITA samples (mean 9.1; sd 16.8; median 3.5). Figure 4 shows that both ruptured and unruptured aneurysm tissue samples contained significantly more bacterial DNA than the LITA control samples (p-values 0.003 and 0.001, respectively).

Both ruptured and unruptured aneurysm tissue samples contained significantly more bacterial DNA than the LITA control samples (p-values 0.003 and 0.001, respectively).

Two separate n-fold values for the amount of bacterial DNA were calculated in each aneurysm sample; the first used the mean amount of bacterial DNA from the LITA samples as a control, and the second used the individual’s own blood sample as a control. The strong correlation between these two values is shown in this figure (r=0.84; p<0.001).

Total bacteria: proportion of the bacterial positive samples; S.mitis –group: Streptococcus mitis –group; S.mitis: Streptococcus mitis; gftP gftG: virulence factors for Streptococcus sanguinis (gftP) and Streptococcus gordonii (gftG); S.anginosus: Streptococcus anginosus;

Staph. sp.: Staphylococcus species; P.intermedia: Prevotella intermedia; P.micra:

Parvimonas micra; P.gingivalis: Porphyromonas gingivalis; A.actinomyc: Aggregatibacter actinomycetemcomitans; F.nucleatum: Fusobacterium nucleatum; D.pneumosintes: Dialister pneumosintes; T.denticola: Treponema denticola

Figure 2. Frequencies of bacterial DNA-positive findings in all cases (42 ruptured and 28 unruptured samples). Tissue samples were taken from aneurysm and the presence of candidate bacteria DNA was assessed using RT-qPCR.

5.2.2 Molecular microbiological findings in aneurysm walls

Using RT-qPCR, bacteria were detected in 49/70 (70%) of the aneurysms (Table 3).

The prevalence of bacteria was 69% in the ruptured aneurysm wall samples and 71%

in the unruptured aneurysm wall samples. In the ruptured group, endodontic and periodontal pathogens were identified in 25/42 (60%) and 18/42 (43%) of aneurysms, respectively. Figure 2 shows the frequencies of bacterial DNA-positive findings in all cases (42 ruptured and 28 unruptured samples) from Studies I and II.

In ruptured aneurysm wall samples, 13 out of 42 (31%) contained the DNA of both periodontal and endodontic bacteria. In the unruptured aneurysm wall samples, 43%

contained DNA from both types of bacteria. Patients with an unruptured aneurysm but with a history of previous SAH had bacterial findings similar to those of the patients without a history of SAH (total positive 71% and 83%, respectively; p=0.62;

Fisher´s test).

Bacterial DNA from the Streptococcus mitis -group and Fusobacterium nucleatum were the most common pathogens. Two separate n-fold values for the amount of bacterial DNA were calculated in each aneurysm sample; the first used the mean amount of bacterial DNA from the LITA samples as a control, and the second used the individual’s own blood sample as a control. Figure 3 shows the strong correlation between these two values (r=0.84; p<0.001). The total amount of bacterial DNA in the aneurysm tissue samples was 12.2-times higher than that found in their control blood samples (mean 12.2; sd 25.9; median 4.1). The amount of bacterial DNA was 9.1 times higher in the aneurysm tissue samples than that found in the LITA samples (mean 9.1; sd 16.8; median 3.5). Figure 4 shows that both ruptured and unruptured aneurysm tissue samples contained significantly more bacterial DNA than the LITA control samples (p-values 0.003 and 0.001, respectively).

Both ruptured and unruptured aneurysm tissue samples contained significantly more bacterial DNA than the LITA control samples (p-values 0.003 and 0.001, respectively).

Two separate n-fold values for the amount of bacterial DNA were calculated in each aneurysm sample; the first used the mean amount of bacterial DNA from the LITA samples as a control, and the second used the individual’s own blood sample as a control. The strong correlation between these two values is shown in this figure (r=0.84; p<0.001).

Total bacteria: proportion of the bacterial positive samples; S.mitis –group: Streptococcus mitis –group; S.mitis: Streptococcus mitis; gftP gftG: virulence factors for Streptococcus sanguinis (gftP) and Streptococcus gordonii (gftG); S.anginosus: Streptococcus anginosus;

Staph. sp.: Staphylococcus species; P.intermedia: Prevotella intermedia; P.micra:

Parvimonas micra; P.gingivalis: Porphyromonas gingivalis; A.actinomyc: Aggregatibacter actinomycetemcomitans; F.nucleatum: Fusobacterium nucleatum; D.pneumosintes: Dialister pneumosintes; T.denticola: Treponema denticola

Figure 2. Frequencies of bacterial DNA-positive findings in all cases (42 ruptured and 28 unruptured samples). Tissue samples were taken from aneurysm and the presence of candidate bacteria DNA was assessed using RT-qPCR.

Figure 3. The correlation between the n-fold values of the bacterial DNA amount (universal primers) using different control samples (i.e., LITA and individual’s own blood).

X-axis represents the n-fold difference between the amounts of total bacterial DNA in the aneurysm sample and in the LITA control sample. Y-axis represents the n-fold difference between the amounts of total bacterial DNA in the aneurysm sample and in the arterial blood sample.

Figure 4. The n-fold difference of total bacterial DNA between the aneurysm tissue samples (ruptured and unruptured) and the control samples (LITA). The amounts of total bacterial DNA were measured using universal primers. Mean value of the total bacterial DNA amounts of the control samples (LITA) was used as a reference level.

Figure 3. The correlation between the n-fold values of the bacterial DNA amount (universal primers) using different control samples (i.e., LITA and individual’s own blood).

X-axis represents the n-fold difference between the amounts of total bacterial DNA in the aneurysm sample and in the LITA control sample. Y-axis represents the n-fold difference between the amounts of total bacterial DNA in the aneurysm sample and in the arterial blood sample.

Figure 4. The n-fold difference of total bacterial DNA between the aneurysm tissue samples (ruptured and unruptured) and the control samples (LITA). The amounts of total bacterial DNA were measured using universal primers. Mean value of the total bacterial DNA amounts of the control samples (LITA) was used as a reference level.