Real time PCR for detection of Chlamydophila pneumoniae in peripheral blood mononuclear cells of patients with multiple sclerosis

Abstract

Sirs: Since the initial report [1] suggesting an association between infection with Chlamydophila pneumoniae (C. pneumoniae) and multiple sclerosis (MS), a possible involvement of this common respiratory pathogen as etiologic agent has been investigated with conflicting results [2–8]. C. pneumoniae is presumed to play a role for its ability to cause a systemic dissemination following respiratory infection, promoting surviving of infected cells of immune system as macrophages, monocytes and lymphocytes. In vivo studies have confirmed that C. pneumoniae disseminates systemically from lungs via peripheral blood mononuclear cells (PBMC) [9, 10]. This event is thought to promote transmigration of monocytes through endothelial cells of brain vessels, resulting in a chronic injury on central nervous system (CNS). A major obstacle for the detection of C. pneumoniae is the lack of a reliable and standardized method. Culture and serology are the traditional methods of isolation of C. pneumoniae, but they suffer from limitations. There are difficulties in growing C. pneumoniae in cell culture, and serology is inadequate in discriminating recent from chronic infections. Lastly, there are a variety of molecular methods available for C. pneumoniae detection but they are in-house assays with different primers, reaction conditions, and DNA extraction procedures. Realtime quantitative PCR offers an attractive alternative to conventional PCR, since it combines amplification and quantitative product detection minimizing the risk of cross-contamination. Previous in vivo studies have suggested that detection of C. pneumoniae DNA by PCR in PBMC is a useful technique for identifying, even at low concentration, C. pneumoniae in the circulation or in the vascular walls in patients with atherosclerotic carotid disease [11, 12]. Up to now, only two studies have investigated the association between C. pneumoniae infection and MS by using conventional PCR in PBMC, reporting contradictory results (rate of detection ranging from 0 to 54%) [4, 13]. Here, we presented the results of the first study using real-time PCR to detect C. pneumoniae DNA in PBMC from MS patients. The cohort consisted of 112 consecutive MS outpatients (88 relapsing-remitting, 17 secondary progressive and 7 primary progressive MS), 39 men and 73 women, regularly attended the S.Andrea MS Centre of the University of Rome ‘‘La Sapienza’’ from January to June 2005. Mean age was 41.1 years (range 21–67), mean disease duration 5.7 years (range 1.5–23) and mean EDSS 3.0 (range 1.0–6.5). Sixty patients were treated with immunomodulatory agents (41 with interferon beta, 5 with glatiramer acetate), 14 patients received immunosuppressive drugs (4 with azathioprine and 10 with mitoxantrone), and 52 patients had no therapy. Whole blood specimens (5 ml) from each patient were processed to isolate PBMC. DNA was extracted by using a Qiagen DNA Mini-kit according to the manufacturer’s instructions. DNA was eluted in a final volume of 50 ll, aliquot, and stored at )20 C. In order to minimize the risk of falsepositive results, negative reagent controls obtained replacing clinical specimens with an equal volume of ultra pure water PCR grade were both included and processed throughout the whole extraction procedure. Detection and quantification of C. pneumoniae DNA were performed by real-time PCR assay, as previously described [14]. Briefly, real-time PCR was based on FRET hybridization probes and LightCycler instrument (Roche Diagnostics, Germany). PCR primers and probes target on an internal, 128 bp region of C. pneumoniae PstI species-specific fragment. All amplification reactions were carried out in a total volume of 10 ll containing 1 ll of extracted DNA. R. Sessa, MD AE G. Schiavoni, MD C. Zagaglia, MD AE M. del Piano, MD Dept. of Public Health Sciences University of Rome ‘‘La Sapienza’’ Rome, Italy