探討台灣地區Aggregatibacter(Actinobacillus) actinomycetemcomitans在牙周炎患者和牙周健康者中的血清型分佈

在過去幾十年的研究中都確認了牙周炎與許多微生物間的密切關係。而在各種類型的牙周炎中，又以局部性侵犯型牙周炎與Aggregatibacter(Actinobacillus) actinomycetemcomitans間的關係最為著名，而且這個牙周致病菌擁有許多的毒性因子。一般認為A.actinomycetemcomitans 的血清型與基因型的分布與牙周狀況是有相關性。目前這個牙周致病菌的血清型被分成六種：a、b、c、d、e、f，而目前的台灣人牙周狀況與A. actinomycetemcomitans 的血清型的關係及此致病菌的血清型分佈情況尚不清楚。因此，我們利用間接免疫螢光法和聚合酶鏈反應來偵測牙周炎患者和牙周健康受試者的牙菌斑，進一步的來分析此致病菌血清型的分佈、盛行率、所佔的比例和牙周狀況的相關性。研究方法：我們收集了45位牙周炎患者(皆為慢性牙周炎)，為高雄醫學大學附設中和醫院牙周病科的門診病患進行說明，採自願參加方式；經由X光片檢查及臨床牙周檢查後，依據American Academy of Periodontology (1999)來分類牙周炎種類。對照組則為自高雄醫學大學牙醫學系學生共45位，受邀自願參加其口腔篩檢，主要為牙醫學系四年級學生為主；經過牙周臨床檢查後，需滿足牙周囊袋≦3 mm和沒有牙齦出血點(bleeding on probing)兩個條件，以便確認為牙周健康者。取其這些受測者口內的牙菌斑，分別以聚合酶鏈反應和免疫螢光法來檢測A. actinomycetemcomitans盛行率、各血清型的出現機率和在牙菌斑中所佔的比例，將所有的資料以Excel軟體建檔，經JMP統計軟體進行連結後，進行X2-test、雙樣本t檢定和ANOVA的統計分析來探討其相關性。結果： 1. 在四十五位慢性牙周炎患者中，A. actinomycetemcomitans的盛行率為33.33%(15/45)，其中血清型a有二位(2/15，13.33%)，血清型b有一位(1/15，6.67%)，血清型c有五位(5/15，33.33%)，血清型d有一位(1/15，6.67%)，血清型e有二位(2/15，13.33%)，不能被分類的血清型共有四位(4/15，26.67%)；在四十五位牙周健康者中的盛行率則為2.22%(1/45)，。 2. 在牙周炎部位中，A. actinomycetemcomitans的盛行率為23.33%(21/90)，血清型出現機率分別為：血清型a為14.29%，血清型b為4.76%，血清型c為23.81%，血清型d為4.76%，血清型e出現機率為14.29%和不能被分類的血清型為38.1%。而其中以不能被分類的血清型為最常見；在牙齦炎部位中，A. actinomycetemcomitans的盛行率為3.03%(1/33)，只有一個樣本出現血清型e；在牙周健康部位中，A. actinomycetemcomitans的盛行率為2.22%(1/45)，只有一個樣本出現不可被分類的血清型。 3. 在牙周炎部位牙齦上牙菌斑中，血清型 a的比例: 0.04%±0.29，血清型 b的比例: 0.03%±0.21，血清型c的比例: 0.06%±0.22，血清型d的比例: 0.03%±0.22，血清型e的比例: 0.02%±0.13；在牙周炎部位的牙齦下牙菌斑中，血清型 a的比例: 0.06%±0.31，血清型 b的比例: 0% ，血清型c的比例: 0.12%±0.38，血清型d的比例: 0%，血清型e的比例: 0.06%±0.30。 4. 在全部九十位受測者中，單一血清型感染機率為75%，混合血清型感染為25%。 5. A. actinomycetemcomitans的感染與性別、年齡分佈和各臨床指數並無相關性。結論：本實驗中主要是以間接免疫螢光法和聚合酶鍊反應來偵測牙周炎患者和牙周健康者中A. actinomycetemcomitans以便探討其盛行率和血清型分佈和感染情況，以建立日後更進一步研究的基礎。在我們的結果中，我們發現A. actinomycetemcomitans的出現在牙周炎患者的機率的確高出牙周健康者許多。由於我們試驗的樣本數太少，因此我們希望日後能有更多探討台灣A. actinomycetemcomitans的研究，以便對這個牙周致病菌更多加了解且對臨床治療上能有幫助。

關鍵字

A. actinomycetemcomitans ；血清型；免疫螢光法；聚合酶鍊反應

並列摘要

Background: In the past several decades researches have been done to investigate the relationship between periodontitis and many microorganisms. Among the many types of periodontitis, the association between localized aggressive periodontitis and Aggregatibacter (Actinobacillus) actinomycetemcomitans had been intensely discussed, as this pathogenic bacteria has many virulent factors. It is commonly recognized that the distributions of A.actinomycetemcomitans serotypes and genotypes are associated with periodontal conditions. The periodontal pathogenic bacteria has been classified into six serotypes: a, b, c, d, e, f. Association between A. actinomycetemcomitans serotypes and periodontal conditions among Taiwanese and the distribution of the serotypes are still unclear. In this study, we used indirect immuno-fluorescence assay and polymerase chain reaction (PCR) to detect dental plaque of periodontal patients and healthy volunteers. The data obtained was used for further analysis of serotype distribution, prevalence, proportion and associations with periodontal status. Methods: Forty-five periodontal patients (all diagnosed chronic periodontitis) from Chung-Ho Memorial Hospital of Kaohsiung Medical University were included in our study. After x-ray and periodontal examinations, each patient’s periodontal type was assigned according to American Academy of Periodontology (1999). The control group consisted of 45 healthy volunteers from Kaohsiung Medical University. The inclusion criteria included: periodontal pockets size of less than or equal to 3 mm and no bleeding on probing. Dental plaques were obtained from the volunteers. The carrier rate, prevalence rate, and serotype distribution of A. actinomycetemcomitans were obtained by using indirect immuno-fluorescenc assay and polymerase chain reaction (PCR). Data collected was analyzed by Excel and JMP, Chi-square test, two-sample t-test and ANOVA were also performed. Result: 1. Among 45 chronic periodontitis patients, the prevalence rate of A. actinomycetemcomitans was 33.33% (15/45). The occurrence of each serotype was: a (13.33%, 2/15), b (6.67%, 1/15), c (33.33%, 5/15), d (6.67%, 1/15), e (13.33%, 2/15), and untypeable (26.67%, 4/15). As for the 45 healthy volunteers, the prevalence rate was 2.22% (1/45), 2. In periodontal sites, the prevalence rate of A. actinomycetemcomitans was 23.33% (21/90). The occurrence of each serotype was: a (14.29%), b (4.76%). c (23.81%), d (4.76%), e (14.29%), and untypeable (38.1%)—the most common type. In gingivitis sites, the prevalence rate was 3.03% (1/33), with only one sample being serotype e. In healthy sites, the prevalence rate was 2.22% (1/45), in which only one sample occurred to untypeable. 3. In the supragingival plaque of periodontitis sites, the occurrence rate of each serotype was: a (0.04%±0.29), b (0.03%±0.21), c (0.06%±0.22), d (0.03%±0.22), e (0.02%±0.13). In the subgingival plaque of periodontitis sites, the occurrence rate of each serotype was: a (0.06%±0.31), b (0%), c (0.12%±0.38), d (0%), e (0.06%±0.30). 4. Among all the 90 patients and volunteers, mono-serotype infection accounted for 75% and poly-serotype infection for 25%. 5. It was concluded that the infection of A. actinomycetemcomitans has no association with sex, age, and other clinical parameters. Conclusions: This study was performed using indirect immuno-fluorescence assay and PCR to detect A. actinomycetemcomitan among periodontal patients and healthy volunteers in order to investigate its prevalence rate, serotype distribution and infection conditions. The results have shown that the occurrence rate of A. actinomycetemcomitan was higher in periodontal patients than healthy volunteers. Further investigations would be done on A. actinomycetemcomitan among Taiwanese people to gain more understanding of this pathogen, and in the hope of improving clinical treatment.