Detection of Chlamydia pneumoniae in Ankylosing Spondylitis Patients

Ankylosing spondylitis is a complex debilitating disease because its pathogenesis is not clear. This study aims at detecting some pathogenesis factors that lead to induce the disease. Chlamydia pneumoniae is one of these pathogenesis factors which acts as a triggering factor for the disease. The study groups included forty Iraqi Ankylosing spondylitis patients and forty healthy persons as a control group. Immunological and molecular examinations were done to detect Chlamydia. pneumoniae in AS group. The immunological results were performed by Enzyme-Linked Immunosorbent Assay (ELISA) to detect anti-IgG and anti-IgM antibodies of C. pneumoniae revealed that five of forty AS patients' samples (12.5%) were positive for antiIgG and IgM C. pneumoniae antibodies compared to controls which revealed seronegative. Molecular detection included 16srRNA and HSP-70 genes were to ensure the serological examination for detection of bacteria in the five blood samples which were positive; therefore, these results improved that C. pneumoniae played a role in the pathogenesis of the disease.


Introduction:
Ankylosing spondylitis (AS) was defined as an inflammatory arthropathies relationship to HLA-B 27 (1), and it is an immune-mediated inflammatory marker, autoantibodies formation was the hallmark of AS (2). There are many infectious agents that are related to AS such as mycobacterial, fungus arthritis (3,4). In addition, recent studies refer to the association between autoimmune diseases and C. pneumoniae bacteria (5). AS disease caused inflammatory back pain which leads to structural and functional impairments (6). Because of the difficulties of C. pneumoniae isolation in vitro; therefore, ELISA and molecular techniques were used to detect the bacteria (7). Recent studies identify C. pneumoniae by DNA amplification of the unique region via using oligonucleotides primers specific for these regions (16srRNA, OMP, and HSP-70 genes) (8,9). While other studies reported the detection of anti-Chlamydia pneumoniae antibodies in the sera of AS patients (10,11). The present study aims to detect the role of C. pneumoniae as a trigger factor for AS.

Studied groups
This study includes forty Iraqi Arab AS patients (30 males and 10 females), who are Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq. E-mail: dunyascience@sc.uobaghdad.edu.iq diagnosed by the rheumatologist consultant of Baghdad Teaching Hospital in Baghdad city, and the matched numbers and gender of healthy persons as a control. The ethics committees of participating universities and the teaching hospital were approved in the study, and the information was obtained from all the studied groups.

Samples collection
Blood samples were collected from September 2016 to December 2016. Five milliliters of venous blood samples were collected from all studied groups, and the blood samples were divided into two parts, three milliliters put in silicon tube, to obtain the serum for the serological examinations, and the residual milliliters put in EDTA tube to isolate the DNA according to the manufacturer manual (Promega, USA) for molecular detection.

C. pneumoniae antibodies detection by ELISA technique
The immunological examination was done by using ELISA technique to detect the anti-C. pneumoniae IgG and IgM antibodies in the sera of studied groups according to the manufacturer leaflet (Human company, Germany).

DNA extraction
The whole DNA was extracted from the EDTA blood samples of the studied groups by using the manufacturer procedure of the commercial kit (Promega company, USA).

Molecular detection of C. pneumoniae
The molecular examination was done by using a polymerase chain reaction (PCR) technique to detect the virulence factors of C. pneumoniae (16srRNA and HSP-70 genes) by using specific primers from Alpha DNA technologies company (Canada) (11) to amplify the target fragments of the chosen genes, PCR products were visualized by electrophoresing in 2% agarose gel (Conda company, USA) which was stained with red stain (Intron Biotechnology Inc., Korea). The size of appeared fragments was compared with a standard DNA ladder fragments size (KAPA™, Universal Ladder KK6302, USA), the primers sequences, PCR conditions, length of PCR products were listed in Table (1).

Results and Discussion:
The results of current study showed that the male: female ratio in AS group was 3:1 in which the age mean ± SE was 40 ± 3.0 years for this group that was compared to healthy control group with age mean ± SE matched to AS group (40 ± 4.3 years). Also, the recent findings appeared that five seropositive sera samples for anti-C. pneumoniae IgG and IgM antibodies (12.5%) of the forty sera samples of AS patients. There were significant differences (P<0.05) between these five seropositive samples (12.5%) and 100% seronegative of the healthy control group (Figure 1). The molecular diagnosis of C. pneumoniae in the blood samples of studied groups shows that these five seropositive of anti-C. pneumoniae IgG and IgM antibodies were positive for detecting the 16sRNA and HSP-70 genes by using specific primers, as shown in Figures 2 and 3, and Table (1). The 16sRNA fragment has a molecular size which was 238 bp, while the HSP-70 fragment has a molecular size 760 bp compared with a DNA ladder. The detection of C. pneumoniae depends on the sensitivity of the diagnostic test because the cell wall structure of bacterium is more complicated. This leads to isolation and diagnosis difficulties (12); therefore, recent studies resort to use the molecular detection besides the immunological detection to ensure the causative and inducer agents of the disease (8,13). The use of 16sRNA gene fragment was to separate between the eukaryotes DNA and prokaryotes DNA, because all the prokaryotes have a 16sRNA gene, while the eukaryotes have a 18sRNA gene (8,14). The analysis of DNA bands results appeared that five samples of the 40 AS group (12.5%) were positive to 16sRNA with molecular size 238 bp compared with the DNA ladder in an agarose gel as shown in Figure (1). This refers to the fact that these samples have the bacteria in the blood of patients. Such, HSP-70 can act as adhesive protein and play a major role in the pathogenesis of C. pneumoniae. It has an association with ribosomal RNA subunit; because the later assumed the stabilization of their structure and HSP-70 has a role in antigen processing and presenting (11). The analysis of DNA band result of HSP-70 revealed that the same seropositive samples of AS group had a positive result to HSP-70 gene fragment when it electrophoresed in agarose gel with molecular size 760 bp compared with the DNA ladder as shown in Figure (2). This result proved that these 5 samples have C. pneumoniae bacteria; because the HSP-70 gene is a specific virulence gene for C. pneumoniae (15). Therefore, these results proved that C. pneumoniae had a role as a triggering factor in the pathogenesis of AS disease by induction mechanism.