Comparison of sample collection methods for the PCR detection of oral anaerobic pathogens

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1 Letters in Applied Microbiology 2003, 36, Comparison of sample collection methods for the PCR detection of oral anaerobic pathogens S.F. Smola 1, G. Rettenberger 1, Th. Simmet 2 and L. Burysek 2 1 Department of Cytogenetics, Gregor Mendel Laboratories for Human Genetics, Wegenerstrasse 15, D Neu-Ulm, Germany, and 2 Department of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, Helmholtzstrasse 20, D Ulm, Germany 2002/208: received 28 June 2002, revised 5 November 2002 and accepted 21 November 2002 ABSTRACT S. F. S M O L A, G. R E T T E N B E R G E R, T H. S I M M E T A N D L. B U R Y S E K Aims: To provide evidence that DNA-PCR diagnostics of oral pathogens based on standard sample collection by paper point insertion from the depth of the periodontal pocket can be replaced by a novel non-invasive collection method based on swab technique from the gingiva. Methods and Results: In this study we compared the results from two collection methods performed in 35 patients with chronic adult periodontitis. Statistical analysis showed a highly significant association of diagnostic results between both collection techniques. Conclusions: The Pocket-out method represents a reliable alternative to the standard collection technique for PCR diagnosis of oral pathogens. Significance and Impact of the Study: Due to its simplicity and non-invasiveness, the Pocket-out collection could be performed in any physician office, or even by the patient himself. With respect to the putative association between periodontal disease and various systemic illnesses, this method could be integrated with various screening programs of oral pathogens. Keywords: collection technique, oral pathogen, PCR diagnostic method, periodontal disease. INTRODUCTION Periodontitis with its various clinical forms represents the most widely distributed type of oral disease. It is an inflammatory condition caused by a chronic bacterial infection with certain anaerobic gram-negative organisms (Armitage 2000a). A growing number of scientific reports point toward a causative link between periodontal disease and various systemic illnesses (Slavkin and Baum 2000). The most severe systemic disorders potentially associated with periodontal disease are endocarditis and other cardiovascular diseases (Scannapieco 1998; Taubert and Dajani 2001), cerebrovascular incidents and ischemia (Kunze et al. 2000) as well as infections of the respiratory system (Terpenning et al. 2001). It is generally accepted that the more severe Correspondence to: L. Burysek, Department of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, Helmholtzstrasse 20, D Ulm, Germany ( ladislav.burysek@medizin.uni-ulm.de). forms of periodontal disease are linked to an increased concentration of specific pathogens, such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Bacteriodes forsythus, Treponema denticola, Fusobacterium nucleatum, and Eikenella corodens. Detection of these anaerobic pathogens by conventional techniques is, however, difficult and time consuming. Recently, PCRbased detection methods gained increasing popularity because of their simplicity, speed, and high sensitivity (Tran and Rudney 1999; Okada et al. 2001; Yuan et al. 2001). Commercially available PCR-based periodontal pathogen detection tests are able to detect specific pathogens with a sensitivity of organisms per collected sample. The suitability of such kits for practical or experimental dental medicine has been repeatedly reported (Smola and Zemen 1999a; Jervoe-Storm et al. 2000; Kleber, 2000; Pfister and Eick 2001). On the background of a causative link between periodontal infections and systemic illnesses, ª 2003 The Society for Applied Microbiology

2 102 S.F. SMOLA ET AL. an increasing interest in oral microbiologic diagnostics could be expected from various disciplines including internal medicine (Armitage 2000b; Emingil et al. 2000; Haraszthy et al. 2000; Slavkin and Baum 2000; Beck et al. 2001; Smola 2001; Terpenning et al. 2001). However, the complexity of the widely used technique for collecting subgingival plaque samples has limited its broad application in routine diagnostics. This technique requires the insertion of sterile endodontic paper points deep into the sulcus/pocket after careful removal of supragingival plaque (Smola and Zemen 1999b; Jervoe-Storm et al. 2000; Kleber 2000; Pfister and Eick 2001), a procedure requiring stomatological practice and equipment. In this study, we compared the pathogen load in samples collected either by the standard technique or by a novel swab technique called Pocket-out. This simple technique requires neither intervention into the gingival pocket nor the removal of supragingival plaque; instead, it collects biologic material from the supragingival area (crest gingiva, attached gingiva, and mucosa). In addition to saliva, cell debris, bacteria, fungi, and viruses, sample material collected by the Pocket-out method also contains genetic material originating from dead periodontal pathogens eluted from the depth of a sulcus/pocket. We demonstrate that the Pocket-out technique provides strikingly similar data to that of the standard subgingival collection method with respect to the detection of pathogens. This study provides evidence that the quantity of pathogens in the periodontal pocket correlates with the number of dead pathogens washed out on the surface of masticatory gingiva. the surface of masticatory mucosa between crest gingiva and the mucogingival border in the range of two teeth, one of which is the collection tooth for paper point method (Fig. 1). In principle, the Pocket-out collection method was strictly applied before standard collection method, in order to exclude possible cross-contamination of the supragingival mucosa by the paper point with living pathogen cells from the depth of the periodontal pocket. All clinical evaluations and collections were made by one periodontist (FS). PCR-based detection of pathogen genomic DNA Immediately after collection, all samples were washed in 1 ml of sterile DNase-free water, and stored at )20 C until analyzed. Total genomic DNA was isolated by a DNA isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) and used as a template for PCR. The pathogen concentration was determined with the commercially available semiquantitative diagnostic kit microdent Ò (Hain Lifescience GmbH, Nehren, Germany). PCR-amplified DNA fragments were hybridized with pathogen-specific sequences linked to the membrane stripes. The amount of hybridized DNA was measured by an enzyme-mediated colorimetric reaction. The color change was then compared against color marker scale divided into five ranges of bacterial counts per sample (< 10 4, , MATERIALS AND METHODS A total of 35 adult patients, years of age, with generalized chronic periodontitis were recruited for the study. Collection methods Standard collection The established technique used for collection of subgingival plaque samples by sterile endodontic paper points (ISO40) (Jervoe-Storm et al. 2000) was used as reference method for sample collection (pocket depth 4 8 mm). To minimize cross-contamination, the collection sites were supragingivally dried before the paper point was inserted into the periodontal pocket (Kleber 2000). Pocket-out collection This method is basically a swab technique with a commercially available foam tip with polymer head (Sterile Foam Tipped Applicator; Hardwood Products Co., Guilford, ME, USA). The swab was performed by applying light pressure with the foam tip on Fig. 1 Scheme of a tooth and gingiva with marked collection areas

3 ORAL PATHOGEN DIAGNOSTICS , , and > ). For clinical diagnosis three levels (number of pathogens per sample) are recognized, negative (< ), slightly positive ( ), and positive (> ). Statistical analysis The results from both standard and Pocket-out collection methods were evaluated by gamma and Kendall s Tau-b measures of association, and by Cohen s weighted kappa coefficient (Ludbrook 2002). All calculations were performed with the SAS software package (SAS Institute Inc., Cary, NC, USA). RESULTS In order to compare the oral pathogen levels in samples collected by standard and Pocket-out methods, collection procedures and diagnostic analysis was performed on 35 volunteers with adult chronic periodontitis. Average values of each pathogen quantities, as measured by PCR detection in samples collected by both methods, were analyzed Negative Slight positive Positive Paper point Pocket-out Aa Pg Pi Bf Td Fig. 2 Pathogen titers per sample (Y-axis) and corresponding diagnostic levels as detected by the paper point and the Pocket-out collection methods. The average findings (mean ± S.E.M) in 35 patients with chronic adult periodontitis are shown for each pathogen tested. Aa, Actinobacillus actinomycetemcomitans; Pg, Porphyromonas gingivalis; Pi, Prevotella intermedia; Bf, Bacteroides forsythus; Td, Treponema denticola (Fig. 2). The lowest number of the pathogen organisms, diagnosed as slightly positive, was detected in the case of A. actinomycetemcomitans where both sampling techniques gave the same result in 34 of 35 (97Æ1%) patients. The average values for all four other pathogens tested were above the positive threshold for both collection methods. The quantities of P. gingivalis and B. forsythus were identical in 32 (91Æ4%) and 31 (88Æ6%) of 35 patients, respectively. The quantities of P. intermedia were identical in 29 (82Æ9%) and T. denticola in 28 (80Æ0%) of 35 patients. The differences detected between results from paper point and Pocket-out techniques were, however, only between two neighboring diagnostic levels; no difference from negative contra positive (or vice versa) was detected. The percentages of agreements for each pathogen are summarized in Table 1. Detailed statistical analyses of results obtained by both methods were performed for each pathogen species separately by three different statistical tests. Measures of association were tested by Kendall s Tau-b and gamma tests in addition to Cohen s weighted kappa analysis. All tests showed a very strong association of the results from both collection methods at the probability level P < 0Æ05, suggesting that paper point and Pocket-out collection methods give similar results (Table 2). Therefore, it is evident that the Pocket-out collection method could represent a truly reliable, non-invasive, and easily performable alternative to standard method in PCR-based detection of periodontal pathogens. Table 2 Statistical analysis of results from the paper point and the Pocket-out collection methods. Kendall s Tau-b and gamma measures of association, as well as weighted kappa analysis were calculated for each pathogen tested, and are shown as mean ± SEM Gamma (%) Kendall s Tau-b (%) Weighted kappa (%) Aa 100Æ0 ±0Æ0 89Æ5 ±9Æ8 92Æ5 ±7Æ7 Pg 100Æ0 ±0Æ0 89Æ3 ±5Æ5 89Æ9 ±5Æ8 Pi 100Æ0 ±0Æ0 86Æ8 ±3Æ9 81Æ6 ±6Æ9 Bf 100Æ0 ±0Æ0 84Æ6 ±6Æ9 83Æ8 ±8Æ0 Td 97Æ0 ±3Æ8 75Æ0 ±8Æ7 69Æ7 ±10Æ3 Table 1 Comparison of the paper point and the Pocket-out collection methods as percentage of agreements (35 ¼ 100%). Differences in one group represent the shift from negative to slightly positive (or vice versa), whereas differences in two groups represent the shift from negative to positive (or vice versa) Aa (%) Pg (%) Pi (%) Bf (%) Td (%) Exact match 100Æ00 92Æ6 85Æ2 92Æ6 81Æ5 Differences in one group 2Æ9 8Æ6 17Æ1 11Æ4 20Æ0 Differences in two groups 0Æ0 0Æ0 0Æ0 0Æ0 0Æ0

4 104 S.F. SMOLA ET AL. DISCUSSION The comparison of two sampling methods for pathogen detections by PCR can be affected by various circumstances. One such factor is the qualitative composition of collected biologic sample. The sulcus liquid (inflammatory exudate) collected by the paper point is often contaminated by aspired blood decreasing the amount of the collected exudate. Moreover, it has been reported that the presence of different blood-derived proteins such as immunoglobulin G, lactoferrin and hemoglobin, has an inhibitory effect on DNA polymerase (Al-Soud and Radstrom 2001). Indeed, for three of five species (A. actinomycetemcomitans, P. intermedia, and T. denticola) we have detected in one subject markedly lower pathogen levels in the sample collected by the paper point method, where the sample was moderately contaminated by blood, than the corresponding sample collected by the Pocket-out method. Another complicated issue is the requirement for sterility during the introduction of the paper point into the periodontal pocket. Even careful drying, removal of the supragingival plaque, and cleaning of the tooth surface does not result in a totally sterile collection environment. Therefore, it is impossible to exclude the possibility of cross-contamination of the paper point by supragingival biologic material, during insertion and removal from the sulcus. The crest gingiva was chosen as the collection site for the Pocket-out method, because this is the critical anatomical location for the contamination of the paper point. Therefore, we cannot exclude the possibility that the bacteria present in the samples collected by the paper point method at least partially originate from the same location as used by the Pocket-out method. It is unrealistic to expect that PCR-DNA diagnostics, detection limit of organisms per sample, could form the basis of clinical decision-making. However, it could inform therapy for patients with cardiovascular disease or diabetes, particularly when prophylaxis is an option. The Pocket-out swab collection technique represents not only a novel non-invasive sampling method for the PCR detection of subgingival anaerobic pathogens in dentistry, but, in combination with other DNA diagnostic methods, it could also represent a new contribution to the sampling techniques required for clinical routine testing as well as for clinical research aiming at the elucidation of possible links between oral pathogens and various systemic illnesses. ACKNOWLEDGEMENTS We are grateful to Dr Mehnert for the overall generous support. We thank Mr Steil and Mrs Glassbrenner for the help with statistical analysis. REFERENCES Al-Soud, W.A. and Radstrom, P. (2001) Purification and characterization of PCR-inhibitory components in blood cells. Journal of Clinical Microbiology 39, Armitage, G.C. 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