Detection of five potentially periodontal pathogenic bacteria in peri ...

Diagnostic Microbiology and Infectious Disease 85 (2016) 289–294

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Detection of five potentially periodontal pathogenic bacteria in peri-implant disease: A comparison of PCR and real-time PCR Gerhard Schmalz a, Sandra Tsigaras b, Sven Rinke c,d, Tanja Kottmann e, Rainer Haak a, Dirk Ziebolz a,⁎ a

Dept. of Cariology, Endodontology and Periodontology, University of Leipzig, Germany Dept. of Preventive Dentistry, Periodontology and Cariology, University Medical Centre Goettingen, Germany Dental Practice Hanau & Alzenau, Germany d Dept. of Prosthodontics, University Medical Centre Goettingen, Germany e Clinical Research Organisation, Hamm, Germany b c

a r t i c l e

i n f o

Article history: Received 10 November 2015 Received in revised form 7 March 2016 Accepted 5 April 2016 Available online 9 April 2016 Keywords: Peri-implant mucositis Peri-implantitis Microbiological findings PCR Real-time PCR

a b s t r a c t The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species. © 2016 Elsevier Inc. All righrs reserved.

1. Introduction In recent years, dental implants for the replacement of missing teeth have exhibited remarkable long-term stability. This assertion is confirmed by current studies that have demonstrated survival and success rates greater than 90% for up to 10 years (Buser et al., 2012; Filippi et al., 2013; Sanz et al., 2015). However, peri-implant complications are still observed; thus, peri-implant mucositis (M) and peri-implantitis (P) remain important biological complications (Mombelli et al., 2012). To avoid these complications and ensure high success rates, early diagnoses are of paramount importance. Both M and P are inflammatory infectious diseases that are primarily caused by bacteria (Mombelli et al., 2012). Accordingly, microbiological analyses of P and M could serve as an important method for early detection. For periodontitis, microbiological testing and monitoring appears to be helpful, especially in patients where the standard therapy is not successful; however, strong evidence for benefit of microbiological testing for periodontal diseases is still missing (Listgarten and Loomer, 2003). Past studies have also investigated peri-implant microbiota (Mombelli and Décaillet, 2011) and reported greater bacterial diversities than those observed in periodontitis biofilms, including staphylococci and enteric bacteria (Belibasakis, 2014; Faveri et al., 2015). However, common periodontal pathogenic ⁎ Corresponding author. Tel.: +49-341-97-21211; fax: +49-341-97-21219. E-mail address: [email protected] (D. Ziebolz). http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.003 0732-8893/© 2016 Elsevier Inc. All righrs reserved.

bacteria are repeatedly detected in peri-implant diseased conditions (Shibli et al., 2008; Máximo et al., 2009; Persson and Renvert, 2014; Albertini et al., 2015). Furthermore, different results regarding the associations of potentially periodontal pathogenic bacteria with M and P are available; thus, the actual opportunities and benefits remain unclear (Mombelli and Décaillet, 2011; Charalampakis and Belibasakis, 2015). Therefore, further investigations appear to be necessary to gain insight into the real microbiological diagnostic opportunities for peri-implant diseases. The potential advantages could include early diagnosis and determination of risk adapted maintenance programs based on microbiological findings, which justifies further investigation of this issue. Different procedures for the microbiological diagnosis of potentially periodontal pathogenic bacteria are available, including polymerase chain reaction (PCR), which has produced exact results in the available studies of periodontitis and peri-implantitis (Haffajee et al., 2009; Ertugrul et al., 2013; Albertini et al., 2015; Eick et al., 2015). With the introduction of commercial tests based on PCR, the qualitative and semi quantitative detection of different bacteria with low temporal expenditure has become possible. An advancement of this process is real-time polymerase chain reaction (RT-PCR), which allows for the quantitative detection of bacterial DNA using fluorescence signals and exhibits high specificity and also successful results regarding the detection of potentially periodontal pathogenic bacteria (Socransky et al., 2005; Boutaga et al., 2007). The available studies have also demonstrated that the bacteria of peri-implant biofilms are detectable with both PCR and RT-PCR

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analyses (Sato et al., 2011; Al-Radha et al., 2012; Galassi et al., 2012; Zhuang et al., 2014; Canullo et al., 2015). However, it is questionable whether conventional PCR provides sufficiently exact results and whether RT-PCR could benefit the microbiological diagnosis of periodontal and peri-implant disease. A recent study compared these methods in terms of periodontitis and found no significant differences, i.e., neither procedure was found to be superior (Eick et al., 2011). Comparable data regarding M and P are already not available, yet. Accordingly, the current study was performed to investigate whether PCR and RT-PCR are able to show differences in detection of five common potentially periodontal pathogenic bacteria between healthy, M and P peri-implant conditions. A further aim was to compare PCR and RT-PCR analyses to determine whether one of these methods is substantially preferable for the microbiological analyses of M and P. The hypothesis of this study was that conventional PCR provides sufficient results for the detection of selected potentially periodontal pathogenic bacteria in peri-implant disease. 2. Methods 2.1. Study design This clinical-experimental examination was performed to investigate different microbiological diagnostic procedures (i.e., PCR and RTPCR) for peri-implant disease. The biofilm samples originated from the patient clientele of a clinical study from this working group (Rinke et al., 2011). The study protocol was reviewed and approved by the ethics committee of the University Medical Center Goettingen, Germany (No. 3/1/09). The patients were informed about the study and provided written informed consent. 2.2. Patients From a pool of 171 peri-implant biofilm samples from 89 patients, 45 were selected for analysis. The implants were classified as healthy or with mucositis or peri-implantitis according the method detailed in a previous publication by this working group (Rinke et al., 2011). Fortyfive samples were randomly divided in the three groups (15 samples in each group) as follows: 15 different samples from healthy implants from implant carriers without further implants in the mouth, 15 different samples from implants with mucositis from implant carriers without further diseased implants in the mouth, and 15 different samples from implants with peri-implantitis with partially samples from additional implants from the same patients. The number of samples investigated conformed to the number of peri-implantitis samples, which were 15 in the patients' clientele. Therefore it was not possible to include more than 15 samples each group. 2.3. Sample collection and DNA isolation Subgingival biofilm samples were collected using sterile paper points. After removing the supragingival biofilm, the implant was drained using cotton rolls. Paper points were placed in the mesial and distal pocket fundi for 10 s, pooled and placed in a transportation tube. For the detection of potentially periodontal pathogenic bacteria, the isolation of DNA is necessary. To achieve this goal, QIAamp DNA Mini Kits (Qiagen, Hilden, Germany) were used according to the manufacturer's instructions. Subsequently, the samples were stored at −20 °C until further processing. 2.4. Polymerase chain reaction (PCR) PCR was performed in the laboratory of the Department of Preventive Dentistry, Periodontology and Cariology of the University Medical Centre Goettingen, Germany. The IDent and Micro-IDent plus (Hain Lifescience, Nehren, Germany) commercial test systems were used for

the qualitative and semiquantitative detections of the potentially periodontal pathogenic bacteria according to manufacturer's protocol. In brief, amplification was performed using a 35-μL mixture of primers and dNTPs (Hain Lifescience, Nehren, Germany), 10.5 μL Mastermix (Qiagen, Hilden, Germany) and 5 μL of the DNA sample or 5 μL of water as a negative control. The cycles were executed in a thermo cycler (Biometra, Goettingen, Germany). Initially, one cycle was conducted for 5 min at 95 °C, subsequently 10 cycles of 30 s at 95 °C and 2 min at 58 °C followed by 20 cycles of 25 s at 95 °C, 40 s at 53 °C and 40 s at 70 °C and a final cycle of 8 min at 70 °C were performed. Hybridization was executed according to the Micro-IDent plus (Hain Lifescience, Nehren, Germany) protocol. In brief, after denaturation and biotinylation, the samples were incubated with hybridization buffer and probes containing membrane strips at 45 °C in a TwinCubator (Hain Lifescience, Nehren, Germany). The membrane strips also contained two control lines (i.e., a conjugate control and an amplification control). Next, highly specific washing steps were executed to remove the nonspecifically bound DNA. Each membrane strip was incubated with a streptavidinalkaline phosphatase complex containing conjugate and intensively washed. The color reactions induced by the reaction between alkaline phosphatase and the substrate on the membrane strips with bound amplification product was registered. Accordingly, the qualitative and semi-quantitative detections of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythia (Tf) and Fusobacterium nucleatum (Fn) were performed based on the colored bands (relative concentrations b103 to N107).

2.5. Real-time PCR Before the RT-PCR was executed, preliminary tests were conducted to detect reliable primers for Aa, Pg, Td, Tf and Fn. Following literature research, whether the primers were able to amplify the DNA of the patient samples was investigated, and water samples simultaneously served as negative controls. The results were validated using the fluorescence signals from real-time PCR and agarose gel electrophoresis. The primers used in this study are listed in Table 1. For exact results, different controls were used. One reference pool containing a mixture of all 45 samples was used as a reference for relative quantification. Moreover, a mixture containing 19 DNA samples with known positive findings for the investigated bacteria from parallel studies was used as a positive reference. Additionally, all samples were analyzed in triplicate. Real-time PCR was performed in a thermocycler (Bio-Rad, Munich, Germany) using 5 μL sample and 20 μL Mastermix containing 12.5 μL SYBRgreen Supermix, 6.5 μL Ampuwa (Fresenius, Bad Homburg, Germany), 0.5 μL sense primer and 0.5 μL antisense primer. Following centrifugation for 3 min at 1500×g, the samples were amplified using the following conditions: initial denaturation for 5 min at 95 °C; 45 cycles of 15 s at 95 °C, 20 s at 60 °C (Aa: 58 °C) and 20 s at 72 °C; a cycle of 1 min at 95 °C and 1 min at 55 °C; and 75 cycles of 10 s at 55 °C. The evaluation of the RT-PCR was executed as a relative quantification in which a pool of 45 probes was set as 100% with a value of 2. Accordingly, the values for the concentrations of bacteria ranged between 0 and 2. Additionally, agarose gel electrophoresis was performed to validate the RT-PCR results. A 3% agarose gel containing 2.4 g agarose (Roth, Karlsruhe, Germany), 80 ml Tris-acetate-EDTA buffer (TAE buffer, 1×, 1:50 with Aqua destillata) was used. Five microliters of each RT-PCR sample was mixed with 1 μL loading buffer (Roth, Karlsruhe, Germany) containing glycerol and bromophenol blue for color detection. One sample containing a pool of 45 samples, single positive and negative control samples, and randomly chosen RT-PCR samples were investigated. Standardized 5 μL 20 bp and 100 bp ladders (Roth, Karlsruhe, Germany) were used as references. The gel electrophoresis was executed in an electrophoresis chamber (Polymehr, Paderborn, Germany) at 85 V for 1 h. Subsequently, the samples were dyed with ethidium


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Table 1 Primer sequences for real-time PCR, which were detected in a preliminary test and used for the analysis. Primer design Aa

forward 5′-GTA CGG CGA AGG TAT TTC CA-3′ backward 5′-CCA ACA ATT CAT CAC GCA AG-3′ forward 5′-GCG ACG CTA TAT GCA AGA CA-3′ backward 5′- GCT GAT GGT GGC ATT ACC TT-3′ forward 5′-TAA TAC CGA ATG TGC TCA TTT ACA T-3′ backward 5′-TCA AAG AAG CAT TCC CTC TTC TTC TTA-3′ MutaGel Periodontitis Kit (Immundiagnostik Bensheim, Germany) forward 5′-CGC AGA AGG TGA AAG TCC TGT AT-3′ backward 5′-TGG TCC TCA CTG ATT CAC ACA GA-3′

Pg Td Tf Fn

Fragment length (bp)

Annealing temperature (°C)

197

58

191

60

316

60

120 23

60 60

Aa = Aggregatibacter actinomycetemcomitans; Pg = Porphyromonas gingivalis; Td = Treponema denticola; Tf = Tanerella forsythia; Fn = Fusobacterium nucleatum.

bromide (Roth, Karlsruhe, Germany) and visualized using a BioDoc Analyzer (Biometra, Goettingen, Germany).

2.6. Statistical analysis The Statistica software (StatSoft, Hamburg, Germany, version 10, 2010) was used for the statistical analyses. Fisher's exact tests were performed (P b 0.05) to detect significant results between the bacteria and disease based on PCR. The significant results between disease and bacteria based on RT-PCR were determined with ANOVA (P b 0.05). Spearman and Kendall Tau tests were used to detect significant correlations between the PCR and RT-PCR results (P b 0.05). Furthermore, sensitivity and specificity was determined for both PCR and RT-PCR results.

3. Results 3.1. PCR analysis All investigated bacteria (i.e., Aa, Pg, Td, Tf and Fn) were detected in the healthy, M and P peri-implant conditions. Detailed concentration and prevalence values are provided in Table 2. The PCR analysis did not reveal any significant results for Aa (P = 0.56), Pg (P = 0.2), Td (P = 0.31), Tf (P = 0.71) and Fn (P = 0.41) regarding healthy (H), mucositis (M) or periimplantitis (P) conditions (Fig. 1a).

3.2. Real-time PCR analysis The RT-PCR analysis detected all investigated potentially periodontal pathogenic bacteria (i.e., Aa, Pg, Td, Tf and Fn) equally in the healthy, M and P peri-implant conditions. Accordingly, no significant results for Aa (P = 0.32), Pg (P = 0.16), Td (P = 0.92), Tf (P = 0.97) or Fn (P = 0.87) with regard to peri-implant disease were identified (Fig. 1b). Detailed concentration and prevalence values are given in Table 2. 3.3. Comparison of PCR and RT-PCR All 45 samples were investigated with both of the compared methods. Both the PCR and RT-PCR analyses detected all investigated potentially periodontal pathogenic bacteria in the healthy, M and P peri-implant conditions (Tables 2 and 3). Regarding prevalence, what means the number of positive findings with regard to the number of investigated samples, PCR and RT-PCR produced similar values for Aa (20% and 22%), Tf (55% and 53%) and Fn (89% and 100%), whereas these methods exhibited greater differences for Pg (67% and 38%) and Td (33% and 55%; Table 4). Exact matches of the concentrations detected by each method were observed for Aa (84%), Pg (33%), Td (51%), Tf (55%) and Fn (29%; Table 4). The correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (Kendall's Tau: 0.49, Spearman: 0.61) and Td (Kendall's Tau: 0.49, Spearman: 0.59). Lower correlations were detected for Aa (Kendall's Tau: 0.38, Spearman: 0.42) and Pg (Kendall's Tau: 0.04, Spearman: 0.04;

Table 2 PCR analysis results for the investigated potentially periodontal pathogenic bacteria. Absolute numbers of samples with the corresponding detection limit is given first and in brackets values are given as % of the 15 investigated samples. The prevalence shows the general number of positive results in relation to the 15 total samples. Concentrations

Under detection limit b10

Aa

Concentrations Pg

Td

Tf

Fn

3

Over detection limit 10

3

Prevalence b10

4

b10

5

N10

6

H M P

13 (86) 11 (73) 12 (80)

1 (7) 1 (7) 2 (13)

1 (7) 0 (0) 0 (0)

0 (0) 0 (0) 0 (0)

0 (0) 3 (20) 1 (7)

2 (13) 4 (27) 3 (20)

H M P H M P H M P H M P

b104 3 (20) 7 (47) 5 (33) 11 (73) 11 (73) 8 (53) 7 (47) 7 (47) 6 (40) 1 (7) 3 (20) 1 (7)

104 5 (33) 2 (13) 0 (0) 3 (20) 1 (7) 4 (27) 5 (33) 2 (13) 4 (27) 1 (7) 2 (13) 1 (7)

b105 2 (13) 0 (0) 1 (7) 1 (7) 0 (0) 1 (7) 0 (0) 3 (20) 1 (7) 10 (67) 4 (27) 6 (40)

b106 2 (13) 2 (13) 5 (33) 0 (0) 3 (20) 1 (7) 2 (13) 3 (20) 3 (20) 3 (20) 5 (33) 7 (47)

N107 3 (20) 4 (27) 4 (2) 0 (0) 0 (0) 1 (7) 1 (7) 0 (0) 1 (7) 0 (0) 1 (7) 0 (0)

12 (80) 8 (53) 10 (67) 4 (27) 4 (27) 7 (47) 8 (53) 8 (53) 9 (60) 14 (93) 12 (80) 14 (93)

H = healthy; M = mucositis; P = peri-implantitis; Aa = Aggregatibacter actinomycetemcomitans; Pg = Porphyromonas gingivalis; Td = Treponema denticola; Tf = Tanerella forsythia; Fn = Fusobacterium nucleatum.

292


A 100 90

prevalence (%)

Table 4 Comparison of PCR and RT-PCR. The prevalences of PCR and RT-PCR are shown as absolute number and in brackets as % in relation to 45 investigated samples. The exact concentration match means the exact match of PCR and RT-PCR. Kendalls Tau and Spearman test results show the correlation between PCR and RT-PCR.

P = 0.41 P = 0.2

80 70

P = 0.71

60

Bacteria

P = 0.31

50

Prevalence RT-PCR [n(%)]

Exact concentration match [n (%)]

Kendall's Tau (R)

Spearman (R)

9 (20) 30 (67) 15 (33) 25 (55) 40 (89)

10 (22) 17 (38) 25 (55) 24 (53) 45 (100)

38 (84) 15 (33) 23 (51) 25 (55) 13 (29)

0.38 0.04 0.49 0.65 0.49

0.42 0.04 0.59 0.78 0.61

healthy mucositis

40 30

Prevalence PCR [n(%)]

P = 0.56

peri-implantitis

20 10 0 Aa

Pg

Td

Tf

Aa Pg Td Tf Fn


Fn

investigated bacteria

B

Table 4). Sensitivity and specificity of PCR and RT-PCR for discrimination between healthy and mucositis as well as healthy and peri-implantitis for the five investigated bacteria is shown in Table 5.

P = 0.87

100

prevalence (%)

90

4. Discussion

80 70

P = 0.92

60

P = 0.97

P = 0.16

50

healthy mucositis

P = 0.32

40

peri-implantitis

30 20 10

4.1. Summary of the main results All investigated potentially periodontal pathogenic bacteria (i.e., Aa, Pg, Td, Tf and Fn) were detected in the healthy, M and P peri-implant conditions by both methods. Significant results for bacteria regarding peri-implant disease were not detected. The PCR–RT-PCR comparison revealed that both methods produced similar results.

0 Aa

Pg

Td

Tf

Fn

4.2. Comparison with the available literature and interpretation of the results

investigated bacteria Fig. 1. a: Prevalence of potentially periodontal pathogenic bacteria based on PCR in healthy, mucositis and peri-implantitis. The P values show the comparison of the bacterial findings using PCR between the three peri-implant conditions and was determined using Fisher-exact test (P b 0.05). b: Prevalence of potentially periodontal pathogenic bacteria by RT-PCR in healthy, mucositis and peri-implantitis. The p-values show the comparison of the bacterial findings using RT-PCR between the three peri-implant conditions and was determined using ANOVA (P b 0.05).

Table 3 RT-PCR analysis results for the investigated potentially periodontal pathogenic bacteria. Absolute numbers of samples with the corresponding detection limit is given first and in brackets values are given as % of the 15 investigated samples. The prevalence shows the general number of positive results in relation to the 15 total samples. Concentrations

Over detection limit Under detection limit 0–≤0.2

Aa

Pg

Td

Tf

Fn

H M P H M P H M P H M P H M P

13 (87) 9 (60) 13 (87) 10 (67) 7 (47) 11 (73) 6 (40) 7 (47) 7 (47) 7 (47) 6 (40) 8 (53) 0 (0) 0 (0) 0 (0)

Prevalence

N0.2–≤0.8 N0.8–≤1.4 N1.4–≤2.0 N2 2 (13) 2 (13) 1 (7) 3 (20) 2 (13) 0 (0) 3 (20) 2 (13) 3 (20) 5 (33) 4 (27) 2 (13) 0 (0) 0 (0) 0 (0)

0 (0) 2 (13) 0 (0) 2 (13) 1 (7) 0 (0) 0 (0) 2 (13) 0 (0) 0 (0) 4 (27) 2 (13) 1 (7) 3 (20) 2 (13)

0 (0) 1 (7) 1 (7) 0 (0) 2 (13) 0 (0) 6 (40) 3 (20) 4 (27) 1 (7) 1 (7) 2 (13) 13 (87) 12 (80) 12 (80)

0 (0) 1 (7) 0 (0) 0 (0) 3 (20) 4 (27) 0 (0) 1 (7) 1 (7) 2 (13) 0 (0) 1 (7) 1 (7) 0 (0) 1 (7)

2 (13) 6 (40) 2 (13) 5 (33) 8 (53) 4 (27) 9 (60) 8 (53) 8 (53) 8 (53) 9 (60) 7 (47) 15 (100) 15 (100) 15 (100)

H = healthy; M = mucositis; P = peri-implantitis; Aa = Aggregatibacter actinomycetemcomitans; Pg = Porphyromonas gingivalis; Td = Treponema denticola; Tf = Tanerella forsythia; Fn = Fusobacterium nucleatum.

In the current study, all detected potentially periodontal pathogenic bacteria were observed with similar prevalences in the healthy, M and P peri-implant conditions. In accordance with these results, several results have been reported that have indicated a lack of reliable differences between healthy and diseased implants in terms of their bacterial populations (Dabdoub et al., 2013; Koyanagi et al., 2013; Zhuang et al., 2014; Canullo et al., 2015). Interestingly, the majority of these studies also performed RT-PCR for detection (Koyanagi et al., 2013; Zhuang et al., 2014; Canullo et al., 2015). In contrast however, different studies are available that have demonstrated a higher prevalence of potentially periodontal pathogenic bacteria in peri-implantitis. A portion of these studies used DNA-DNA checkerboard hybridization as the detection method (Shibli et al., 2008; Máximo et al., 2009; Persson and Renvert, 2014). Additional studies have performed PCR for the detection of potentially periodontal pathogenic bacteria in peri-implant disease Table 5 Results of sensitivity and specificity analysis for PCR and RT-PCR. Sensitivity and specificity for discrimination between healthy and mucositis as well as healthy and peri-implantitis was determined for the five investigated bacteria. Method

Bacteria

PCR

Aa Pg Td Tf Fn Aa Pg Td Tf Fn

RT-PCR

Healthy vs mucositis

Healthy vs. peri-implantititis

Sensitivity

Specificity

Sensitivity

Specificity

40% 53% 53% 60% 100% 27% 53% 27% 53% 80%

87% 67% 40% 47% 100% 87% 20% 73% 47% 7%

13% 27% 53% 47% 100% 20% 67% 47% 60% 93%

87% 67% 40% 47% 100% 87% 20% 27% 47% 7%



(Albertini et al., 2015; Eick et al., 2015). In this context, Pg, Tf, and Td have been detected, whereas Aa was not found in P (Albertini et al., 2015). Moreover, Eick et al. (2015) reported associations of Td with PPD and inflammation (BOP and GI) on implants (Eick et al., 2015). This was not confirmed in the PCR analysis of the current study. In addition to potentially periodontal pathogenic bacteria, many studies have detected additional microbiota that are potentially associated with P among which Staphylococcus ssp. might be the most common (Shibli et al., 2008; Máximo et al., 2009; Persson and Renvert, 2014; Eick et al., 2015). However, the potential roles of these microbes remain unclear (Belibasakis, 2014). As presented in the literature, the findings related to peri-implant biofilms are heterogeneous, and the real potential is not completely clear (Faveri et al., 2015; Charalampakis and Belibasakis, 2015). For this reason, the current study focused on common potentially periodontal pathogenic bacteria. Therefore, in relation to Socransky et al, Pg, Td and Tf were chosen as major pathogenes for periodontal diseases (Socransky et al., 1998). Additionally, Aa which might be associated to aggressive periodontal diseases (Könönen and Müller, 2014) and Fn as a ‘bridge-organism’ with an important role in formation of potentially periodontal pathogenic biofilm (Sharma et al., 2005) were investigated. All of these bacteria were already detected in peri-implant diseases; however, results were inconsistent (Shibli et al., 2008; Máximo et al., 2009; Persson and Renvert, 2014; Albertini et al., 2015). However, in the interpretation of the results of the current study, the facts that only five potentially periodontal pathogenic bacteria were investigated in only 45 samples must be taken into account. In comparable studies, the sample sizes have ranged between 44 and 504 periimplant samples (Shibli et al., 2008; Eick et al., 2015). Nevertheless, detections of all investigated potentially periodontal pathogenic bacteria in the healthy, P and M peri-implant conditions casts doubt on the diagnostic benefit. Consequently, based on the current literature and the results of the present study, there appears to be no clear advantage of microbiological testing for peri-implant disease at the moment. An additional aspect is the comparison of PCR and RT-PCR in the detection of potentially periodontal pathogenic bacteria in M and P. Conventional PCR, particularly that using Micro-IDent plus (Hain Lifescience, Nehren, Germany) has been repeatedly and successfully performed in periodontal and peri-implant disease in the available studies and is therefore thought to be a valid method (Haffajee et al., 2009; Ertugrul et al., 2013; Eick et al., 2015). Moreover, RT-PCR allows for the effective detection of potentially periodontal pathogenic bacteria as demonstrated in recent investigations (Sato et al., 2011; Galassi et al., 2012; Zhuang et al., 2014; Canullo et al., 2015). The current study revealed a very low correlation between PCR and RT-PCR for Pg (0.04). PCR revealed a greater prevalence of Pg, particularly in the healthy peri-implant conditions. Furthermore, Pg (33%) and Fn (29%) exhibited much weaker matches than Aa (84%), Td (51%) and Tf (55%). Different methodological reasons could potentially be responsible for these results. For example, the RT-PCR was performed entirely in a thermocycler, whereas the PCR was partly performed in a shaking bath and was thus associated with a greater risk of contamination. Additionally, the PCR primers used in these tests have not been undisclosed by the manufacturer, whereas the RT-PCR primers were designed based on literature research (Table 1) and validated in a preliminary experiment. Consequently, these methodological differences might be responsible for the differences between the methods. To the authors' knowledge, there is no available study that has compared these methods in terms of peri-implant disease. Regarding periodontitis, one investigation demonstrated significant correlations between both methods for Aa (0.68), Pg (0.74), Td (0.62), Tf (0.69) and Fn (0.59) (Eick et al., 2011). Eick et al. (2011) also concluded that conventional PCR is more suitable and useful for dental practice. These results are in accordance with the results of the current study, particularly in terms of the similar observed correlations between PCR and RT-PCR for Tf (0.78), Fn (0.61) and Td (0.59). The correlation

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coefficients for Aa (0.42) and Pg (0.04) observed in the current study were clearly lower than those reported by Eick et al. (2011). These differences might have been caused by the use of different primers. Additional studies comparing PCR and RT-PCR are not available for either peri-implantitis or periodontitis. However, several studies have investigated PCR or RT-PCR compared with checkerboard hybridization or cultivation; these studies demonstrated that PCR is the predominant method (Riggio et al., 1996; Eick and Pfister, 2002; Haffajee et al., 2009). Furthermore, a meta-analysis compared the detections of Aa and Pg by RT-PCR and cultivation and found that RT-PCR is more exact (Atieh, 2008). In contrast, Lau et al. (2004) reported a good match between RT-PCR and cultivation (Lau et al., 2004). Therefore, the method should be chosen according to the aim of the investigation. RT-PCR ensures the fast quantification of potentially periodontal pathogenic bacteria, whereas antibiogram cultivation is necessary for the detection of bacterial activity (Verner et al., 2006). For the semi-quantitative detection of microbiota in dental practice, commercial test systems based on PCR appear to be sufficient. Accordingly, our hypothesis that conventional PCR provides sufficient results the diagnoses of peri-implant diseases is confirmed. Nevertheless, the results depend on the technique and test system and should be interpreted only with consideration for the clinical diagnosis (Untch and Schlagenhauf, 2015). Taking the results for analysis of sensitivity and specificity into account, the benefit of both PCR and RTPCR appears questionable. Heterogeneous and low values indicate an insufficient distinctness between healthy and diseased peri-implant conditions for both methods. For periodontal diseases, Eick et al., 2011 found higher sensitivity and specificity. Therefore, the benefit of investigation of potentially periodontal pathogenic bacteria in periodontal diseases appears to be higher compared to peri-implant diseases. 4.3. Strengths and limitations To the authors' knowledge, this is the first study to compare PCR and RT-PCR in the detection of peri-implant disease. Furthermore, the detailed analyses of the potentially periodontal pathogenic bacteria in the healthy, M and P peri-implant conditions are a strength of this study. The study was limited by small sample size of 45. However, smaller sample sizes have been used to detect potentially periodontal pathogenic bacteria; so e.g., De Leitão et al. (2005) investigated 19 samples (De Leitão et al., 2005). The investigation of more than one sample of patients with P was also suboptimal; however, the patient clientele consisted of only very few patients with P. Furthermore, methodological limitations regarding diagnosis and sample collection must be mentioned. The clinically diagnosis of H and M sites could be a source of flaws; however the diagnosis was executed with highest care. Similarly, the sample collection in H and M sites is difficult, what should be considered in the interpretation of results. The use of different primers may also have negatively influenced the correlation results. Especially, the primer sequences of the commercial test systems IDent and Micro-IDent plus (Hain Lifescience, Nehren, Germany) are not given by the manufacturer. This must be taken into account in interpretation of the results. A solution for future studies could be the use of arbitrarily primed PCR (AP-PCR) (Ito, 2010). With this method, the identification of exact bacterial genotypes the accordingly specific primer design might ensure better comparability. An additional strength of this study is that the same sample was investigated with both methods. The performance of the experiments in triplicate and the preliminary test also increased the quality of the current study. 5. Conclusion Within the limitations of the current study, the similar PCR and RTPCR results suggest that neither method is superior. Accordingly, PCRbased test systems are sufficient for dental practice. The detection of similar levels of potentially periodontal pathogenic bacteria in the

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healthy, M and P peri-implant conditions makes the advantages of microbiological testing for peri-implant disease questionable. Based on microbiological findings of potentially periodontal pathogenic bacteria it is impossible to diagnose peri-implant diseases. Clinical diagnostic of M and P remain indispensable. Acknowledgments The authors thank Mrs. M. Hoch for support in the laboratory microbiological analysis. Conflict of interest and source of funding statement The authors declare that they have no financial or other relationships that might lead to a conflict of interest. References Albertini M, López-Cerero L, O'Sullivan MG, Chereguini CF, Ballesta S, Ríos V, et al. Assessment of periodontal and opportunistic flora in patients with peri-implantitis. Clin Oral Implants Res 2015;26:937–41. Al-Radha ASD, Pal A, Pettemerides AP, Jenkinson HF. Molecular analysis of microbiota associated with peri-implant diseases. J Dent 2012;40:989–98. Atieh MA. Accuracy of real-time polymerase chain reaction versus anaerobic culture in detection of aggregatibacter actinomycetemcomitans and porphyromonas gingivalis: a meta-analysis. J Periodontol 2008;79:1620–9. Belibasakis GN. Microbiological and immuno-pathological aspects of peri-implant diseases. Arch Oral Biol 2014;59:66–72. Boutaga K, Savelkoul PHM, Winkel EG, Van Winkelhoff AJ. Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction. J Periodontol 2007;78:79–86. Buser D, Janner SF, Wittneben JG, Brägger U, Ramseier CA, Salvi GE. 10-year survival and success rates of 511 titanium implants with a sandblasted and acid-etched surface: a retrospective study in 303 partially edentulous patients. Clin Implant Dent Relat Res 2012;14:839–51. Canullo L, Peñarrocha-Oltra D, Covani U, Botticelli D, Serino G, Penarrocha M. Clinical and microbiological findings in patients with peri-implantitis: a cross-sectional study. Clin Oral Implants Res 2015;26. http://dx.doi.org/10.1111/clr.12557. [Epub ahead of print]. Charalampakis G, Belibasakis GN. Microbiome of peri-implant infections: lessons from conventional, molecular and metagenomic analyses. Virulence 2015;6:183–7. Dabdoub SM, Tsigarida AA, Kumar PS. Patient-specific analysis of periodontal and periimplant microbiomes. J Dent Res 2013;92:168–75. De Leitão JAO, De Lorenzo JL, Avila-Campos MJ, Sendyk WR. Analysis of the presence of pathogens which predict the risk of disease at peri-implant sites through polymerase chain reaction (PCR). Braz Oral Res 2005;19:52–7. Eick S, Pfister W. Comparison of microbial cultivation and a commercial PCR based method for detection of periodontopathogenic species in subgingival plaque samples. J Clin Periodontol 2002;29:638–44. Eick S, Straube A, Guentsch A, Pfister W, Jentsch H. Comparison of real-time polymerase chain reaction and DNA-strip technology in microbiological evaluation of periodontitis treatment. Diagn Microbiol Infect Dis 2011;69:12–20. Eick S, Ramseier CA, Rothenberger K, Brägger U, Buser D, Salvi GE. Microbiota at teeth and implants in partially edentulous patients. A 10-year retrospective study. Clin Oral Implants Res 2015. http://dx.doi.org/10.1111/clr.12588. [Epub ahead of print]. Ertugrul AS, Arslan U, Dursun R, Hakki SS. Periodontopathogen profile of healthy and oral lichen planus patients with gingivitis or periodontitis. Int J Oral Sci 2013;5:92–7. Faveri M, Figueiredo LC, Shibli JA, Pérez-Chaparro PJ, Feres M. Microbiological diversity of peri-implantitis biofilms. Adv Exp Med Biol 2015;830:85–96. Filippi A, Higginbottom FL, Lambrecht T, Levin BP, Meier JL, Rosen PS, et al. A prospective noninterventional study todocument implant success and survival of the Straumann

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