Investigation of Chlamydia pneumoniae Infection in Patients With Multiple Sclerosis: A Case-Control Study

Background: Chlamydia pneumonia has currently been proposed to be involved in multiple sclerosis (MS) pathogenesis. The aim of this study was to evaluate the serum level of anti-C. pneumoniae immunoglobulin G (IgG) in patients with MS compared to the control group, which may be helpful in further understanding of MS etiology. Methods: The serum was obtained from 66 people with MS and 20 healthy people as patient and control groups, respectively. Serums were matched to the patient’s age, gender, and place of residence. Finally, anti-C. pneumoniae IgG was measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Results: Sixty-six patients (25 men and 41 women within the age range of 17-56 years and mean ± SD of 35.53 ± 9.56) with MS and 20 control subjects (8 men and 12 women with the age range of 25-56 and mean ± SD of 38.3 ± 9.66) were enrolled in this study. The mean serum anti-C. pneumoniae IgG of patients with MS (28.82 ± 33.54 RU/mL) was compared with that of the control group (4.075 ± 6.691 RU/mL) and a statistically significant difference was observed in this regard (P < 0.001). Conclusions: In general, there was a significant relationship between the incidence of MS and the C. pneumoniae infection. However, the actual involvement of C. pneumoniae in MS is still a matter of debate and needs to be clarified in further studies (e.g., by creating animal models and conducting trial tests).


Methods
This study included 66 patients with MS who were admitted in the Neurology Department at Farshchian hospital of Hamadan province, Iran between December 2017 and August 2018. This disease was diagnosed by specialized neurologists based on McDonald's criteria (21) and 20 healthy people were matched with patients' age, gender, and place of residence.
Three milliliters of blood samples were collected from all individuals in the study and then were centrifuged at 3000 rpm for 7 minutes. The sera were stored at -70°C until further analysis. Then, C. pneumoniae IgG was measured by the ELISA kit (Euroimmun, Germany) according to the manufacturer's instructions.

Statistical Analysis
Data analysis was performed using SPSS software, version 16. P values less than 0.05 were considered statistically significant.

Results
In general, 66 patients (25 men and 41 women in the age range of 17-56 years and with the mean ± SD of 35.53 ± 9.56) with MS and 20 control subjects (8 men and 12 women within the age range of 25-56 and the mean ± SD of 38.3 ± 9.66) were enrolled in this study. Table 1 presents the anti-C. pneumoniae IgG and demographic characteristics in patients with MS and healthy subjects.
The mean serum anti-C. pneumoniae IgG of patients with MS (28.82 ± 33.54 RU/mL) was compared with that of the control group (6.69 ± 4.07 RU/mL) indicating a statistically significant difference (P < 0.001).

Discussion
MS is considered a chronic inflammatory disease that affects the CNS and causes several neurological disorders (22). Thus, this study investigated the relationship between MS and C. pneumoniae infection in a sample of the Iranian population. Although no evidence is available regarding the main cause of the disease, some epidemiologic studies confirmed the role of genetic factors and the environment in the development of the disease (23)(24)(25)(26). Therefore, the role of infectious agents as environmental factors is important from this perspective (11). C. pneumoniae is one of the most important organisms and its role has been recently emphasized in MS. Due to the inability of the host to complete the elimination of C. pneumoniae, this bacterium can lead to persistent infections (27).
Monocytes pass the bacterium from the blood-brain barrier and enter it into the CNS, which ultimately causes inflammation. Therefore, it can play a role in the development of MS (28). Several studies have confirmed the association between MS and C. pneumoniae infection. In this study, it was found that anti-C. pneumoniae IgG in MS patients was significantly higher compared to the control group. The number of patients and the control group in this study was 66 (25 men and 41 women) and 20 (8 men and 12 women), respectively. In addition, the mean age of the patient and control groups was 35.53 and 38.3, respectively. In contrast to our study, Ramroodi et al showed that there is no direct correlation between MS and C. pneumoniae infection. In this study, the number of patients and healthy people was 80 and their average age was 36.63 and 36.53, respectively. Further, the mean age was similar to that of our study (29). In another study, Khaliel and Abbas also found no significant relationship between C. pneumoniae infection and MS disease. The mean age of people with MS and healthy people was 34.5 and 34.7, respectively, and the number of patient and control groups was 60 (25 men and 35 women) and 30 (8 men and 22 women), respectively (30). Similarly, Aghaei et al demonstrated no association between MS and C. pneumoniae in Iranian people. One reason for the discrepancy between the findings of this study and those of our study could be the difference in geographical areas. The number of patients and the control group in that study was 85 (18 men and 67 women) and 50 (12 men and 38 women), respectively, and their mean age was reported to be 33.8 and 33.9. Some studies (30)(31) were similar to our study in that the number of people in the control group was more tham that in the patients group (31). Munger et al found that there was a positive association between C. pneumoniae infection  (35). Currently, C. pneumoniae has been proposed to be involved in the pathogenesis of MS, and thus more studies with higher sample sizes are needed to further demonstrate the role of C. pneumoniae in the development of MS. However, the actual involvement of C. pneumoniae in MS is still a matter of debate and needs further clarification in future studies (e.g., by the creation of animal models.)