High prevalence of Chlamydia pneumoniae infection in cyclosporin A-induced post-transplant gingival overgrowth tissue and evidence for the possibility of persistent infection despite short-term treatment with azithromycin

Harald C. Worm, Gerhard H. Wirnsberger, Astrid Mauric and Herwig Holzer

Division of Nephrology, Department of Internal Medicine, Karl-Franzens University, Graz, Austria

Correspondence and offprint requests to: Harald Worm, MD, Division of Nephrology, Department of Internal Medicine, Karl-Franzens University, Auenbruggerplatz 15, A-8036 Graz, Austria. Email: harald.worm{at}klinikum-graz.at



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Cyclosporin A (CsA) induces gingival overgrowth (GO) in up to a quarter of CsA-treated renal transplant recipients. A short-term therapy with azithromycin effectively reduces GO, indicating a possible involvement of microorganisms in the pathogenesis of CsA-induced GO. We aimed to determine if there could be any relationship between infection with Chlamydia pneumoniae and GO pathogenesis. In addition, we determined the long-term persistence rate of C. pneumoniae infection in residual GO tissue when azithromycin treatment failed to eliminate GO.

Methods. Chlamydia pneumoniae IgG and IgM antibody titres were measured by microimmunofluorescence technique in sera of kidney recipients with (n = 11) and without (n = 89) GO. GOs were rated and gingivectomies were performed before treatment with 500 mg of azithromycin for 3 days and at months 6 and 12 post-treatment when C. pneumoniae titres were re-evaluated. Nested polymerase chain reaction was performed to identify C. pneumoniae-specific DNA in GO tissues. Results of C. pneumoniae antibody titres from patients with GO were compared with pair-matched controls without GO.

Results. Chlamydia pneumoniae IgM titres were elevated in five of 11 patients with GO and in none without GO, whereas the difference of C. pneumoniae IgG titres between patients with GO and pair-matched controls did not reach significance (P<0.57). Chlamydia pneumoniae-specific DNA was found in 10 of 11 GO tissue samples pre-treatment. Azithromycin therapy effectively reduced GO and C. pneumoniae IgM titres. In residual GO, C. pneumoniae-specific DNA remained detectable after 1 year in all GO tissue samples despite azithromycin treatment. The C.pneumoniae IgM titres correlated with GO scores.

Conclusion. Chlamydia pneumoniae infection is highly prevalent in CsA-induced GO. The infection can persist over a long period in residual GO despite short-term azithromycin therapy. The results indicate that CsA immunosuppression enhances C. pneumoniae infection rates in non-cardiovascular tissue.

Keywords: atherosclerosis; Chlamydia pneumoniae; cyclosporin A; gingival overgrowth; renal transplantation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Post-transplant gingival overgrowth (GO) is a significant complication of cyclosporin A (CsA)-based immunosuppressive drug regimens, affecting up to a quarter of CsA-treated renal transplant recipients [1]. In severe cases of gingival enlargement, gingivectomy and gingivoplasty are occasionally necessary. Histological examination of such enlarged gingival tissue shows signs of acute or chronic inflammation [2]. However, the mechanism of CsA-induced GO is unknown. Azithromycin, an azalid antimicrobacterial agent, has been shown to reduce GO effectively [36]. Intracellular concentrations of azithromycin may reach up to 200-fold those of serum concentrations [7]. Therefore, azithromycin is a mainstay treatment option for intracellular pathogens such as Chlamydia pneumoniae. The purpose of our study was to determine the infection rate of GO tissue with C. pneumoniae in a prospectively studied consecutive series of CsA-treated kidney transplant recipients requiring gingivectomy. These findings were compared with serology in kidney transplant recipients with and without GO to determine a possible relationship between C. pneumoniae infection and GO pathogenesis. In addition, we studied the efficacy of azithromycin to reduce GO and C. pneumoniae infection rates and determined if C. pneumoniae could persist in residual GO tissue when azithromycin treatment failed to eliminate GO.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The study population consisted of renal transplant recipients all receiving a basic two drug immunosuppression including CsA and prednisone. Established medications remained unchanged throughout the study. CsA blood levels were monitored routinely and the dosage adapted if necessary to be in a range between 100 and 150 ng/ml trough level. First time requirement of Ca-channel blockers or phenytoin during the study period was defined as an exclusion criterion. At baseline, serum samples were collected from patients with (n = 11) and without GO (n = 89) and tested for C. pneumoniae IgG and IgM antibodies. Patients with GO underwent gingivectomy performed by a dental surgeon and were subsequently treated with 500 mg of azithromycin for 3 days. GO was rated according to a previously published classification scheme (Table 1) [1]. Chlamydia pneumoniae IgG antibody titres from patients with GO were pair-matched with results from patients of the same sex, and similar age and duration of immunosuppression without GO (Table 1). Statistical analysis was performed using Prophet Statistics Version 6.0 (AB Tech. Corp., Virginia). A match-pair analysis for categoric variables was performed for all cases and controls using Mantel–Haenzel {chi}2 for multiple controls. In the post-treatment phase, C. pneumoniae antibody titres and the presence of GO were re-evaluated at months 6 and 12, and patients with residual GO were again assigned to gingivectomy (Table 2). Gingivectomied tissue samples were studied for the presence of C. pneumoniae-specific DNA sequences using a nested polymerase chain reaction (PCR) assay (Figure 1).


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Table 1. PCR results, scores of gingival overgrowth (GO) and C. pneumoniae IgM/IgG antibody titres at baseline in kidney transplant recipients with CsA-induced GO and C. pneumoniae IgG antibody titres in pair-matched controls (C. pneumoniae IgM titres were negative for controls)

 

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Table 2. Follow-up data of kidney transplant recipients with CsA-induced GO over a 12-month observation period subsequent to a 3-day course of azithromycin therapy

 


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Fig. 1. Electrophoreses of PCR products: typical profile of amplified C.pneumoniae DNA (arrow: 438 bp). Lane numbers correspond to patient numbers (11 showed no visible band); M, 100 bp DNA molecular weight ladder; C, PCR negative control (skin fibroblasts).

 
Serology
Sera were prepared within 2 h after collection and stored at –80°C in 2 ml aliquots. The C. pneumoniae IgG and IgM antibody titres were tested using a commercial microimmunofluorescence test (MIKRO-IFT, Labsystems OY, Finland). IgM-positive sera (titre ≥1:16) were retested after removal of the rheumatoid factor. IgG titres <1:64 were classified as ‘probably negative’; ‘high positive’ IgG titres (≥1:64) were rated as chronic, or past infections in the absence of a positive IgM titre, and ‘high positive’ IgG titres combined with positive IgM titres were considered indicative of acute infection (adapted from Schumacher et al. [8]).

Molecular tests
A total of 18 tissue samples (11 at baseline, four at 6 months and three at 12 months) were collected in buffered formalin. All specimens underwent light microscopic examinations by a pathologist and PCR testing using a C. pneumoniae-specific nested PCR protocol, as described previously [9]. All specimens were prepared using stringent precautions in a sterile bench. For control purposes, samples from skin fibroblasts were included in each preparation batch. DNA was detected following a previously published method [10] using an HL-1/HR-1 primer pair that resulted in a 438 bp C.pneumoniae target sequence of unknown function. All PCR products were visualized in an argarose gel with added ethidium bromide on a UV transilluminator. For confirmation, DNA hybridization was performed with a non-radioactive probe (oligonucleotide HM-1 3'-tailed with digoxigenin-11-dUTP/dATE) followed by a histochemical visualization after incubation with alkaline phosphatase-coupled anti-digoxigenin antibody (Roche).



   Results
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
IgM antibodies to C. pneumoniae were positive in five of 11 (45%) CsA-treated patients with GO and in none without GO. All five patients with positive IgM titres had score 3 GO (Table 1). Elevated IgG antibodies to C. pneumoniae were found in seven of 11 (64%) patients with GO and in 46 out of 89 (52%) without GO; however, differences of IgG titres did not reach significance (P<0.57) between patients with GO and pair-matched controls. Chlamydia pneumoniae-specific DNA was detected in 10 out of 11 gingivectomy tissue samples at baseline. The single PCR-negative GO tissue sample was obtained from a patient negative for C. pneumoniae IgM antibodies. In the sequel of azithromycin therapy, C. pneumoniae IgG titres remained at unchanged levels in patients with GO during the 12 months' observation time (data not shown). In all patients, gingival bleeding stopped within the first week (mean 3.9 days) after antibiotic exposure. Gingival overgrowth improved after a lapse of 1 month, and hyperplasia scores decreased steadily, indicating a significant net reduction of GO (Table 2). Histological examinations demonstrated chronic inflammation in all specimens with poor cellular and richly collagenous fibrous connective tissue. At the end of the study, GO had completely resolved in six patients, and three patients had GO score 1. All three patients with residual score 1 GO had a substantial net reduction of GO compared with baseline scores. Only two patients had a relapse at the end of the study with GO scores 2 and 3, respectively, with mild gingival bleeding. Both patients had high C. pneumoniae IgM titres (1:32) at that time, suggesting a close correlation between IgM titres and GO (Table 2). Chlamydia pneumoniae-specific DNA was detected in all four gingivectomy tissue samples at 6 months, and in all three at 12 months (two patients with GO score 1 refused gingivectomy at 12 months); whereas in two added controls (skin fibroblasts), C. pneumoniae-specific DNA was not detectable (Figure 1).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Our findings demonstrate a high (nearly complete) infection rate of GO tissue with C. pneumoniae in kidney transplant recipients on CsA medication. Chlamydia pneumoniae infection of GO tissue was correlated to elevated C. pneumoniae IgM titres, but no such statistically significant correlation was seen if only C. pneumoniae IgG titres were elevated. GO, therefore, seems to be associated with acute, or ‘active’ C. pneumoniae infection, respectively. ‘In vitro’ studies suggested that C. pneumoniae is taken up both by non-phagocytic cells (i.e. endothelial cells or fibroblasts) and by phagocytes [11,12]. However, in tissue specimens, C. pneumoniae has been detected predominantly in atherosclerotic plaques and only infrequently in non-cardiovascular tissue [13,14]. Our findings suggest that patients on CsA medication might be at higher risk for persistent C. pneumoniae infection of non-cardiovascular tissue.

The question arises of whether C. pneumoniae could, at least in part, be the causal agent of gingival inflammation. Recently, it was reported that C. pneumoniae increases the proliferation of murine fibroblasts by activating p44/p42 mitogen-activated protein kinase [12]. CsA medication, on the other hand, was shown to impair the control of C. pneumoniae infection in a dose-dependent manner by reducing the expression of several critical cytokine genes that promote T-cell activation [15]. In addition, CsA upregulated proinflammatory cytokines and growth factors (such as transforming growth factor-ß) in gingival tissue [1618]. Combined with C. pneumoniae-induced increased proliferation of fibroblasts, this could lead to inflammation and hyperplasia of gingival tissue. The effectiveness of azithromycin therapy in reducing GO further supports the hypothesis that C. pneumoniae infection is directly involved in the pathogenesis of GO. According to recent reports [36], we could demonstrate that short-term azithromycin therapy effectively reduces GO. Likewise, C. pneumoniae antibodies declined in the sequel of azithromycin therapy. Only two patients had grade 2 or 3 GO at a 1 year follow-up control and only these two patients had elevated C. pneumoniae IgM titres, suggesting ongoing (‘active’) C. pneumoniae infection. In all samples of residual GO tissues obtained after 6 and 12 months, C. pneumoniae infection remained detectable. Further studies are needed to determine if a longer duration of azithromycin therapy could be of benefit for such patients. Additional health hazards could arise from persistent C. pneumoniae infection for patients on CsA medication. Serostudies indicated a possible association of persistent C. pneumoniae infection with atherosclerosis, acute myocardial infarction, cerebrovascular disease, hypertension or wheezing [1922]. Seropositivity for C. pneumoniae infection was associated with reduced survival in haemodialysis patients and patients on peritoneal dialysis, due to cardiovascular events [23,24]. Similar implications of C. pneumoniae infection need further evaluation for transplant recipients on CsA immunosuppression, especially for those with GO. However, our study has several limitations. The sample size of analysed GO tissues is relatively small and we have not studied GO tissues of patients without CsA medication. Therefore, sample bias cannot be excluded. In summary, our results demonstrate a high, nearly complete, infection rate of GO tissue with C. pneumoniae. Short-term azithromycin therapy effectively reduces GO scores and C. pneumoniae IgM titres. In residual GO tissue, C. pneumoniae remains detectable, demonstrating the persistence of C. pneumoniae infection.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 16. 7.03
Accepted in revised form: 9.12.03