Actinobacillus actinomycetemcomitans in Families Afflicted With Periodontitis John C. Gunsolley, * Richard R. and Harvey . Schenkein*
The purpose
Ranney,f Joseph J. Zambón,# John A. Burmeister,
*
determine the prevalence of Actinobacillus actinoin individuals from families where at least one individual has an mycetemcomitans (Aa) form of onset Periodontitis. Twenty-three families with 73 subjects were evaluated early in this study. Forty-seven early onset Periodontitis subjects outside the 23 families were also studied. Prevalence of detection of Aa in family members ranged from 49% to 66% among groups constituted by clinical findings indicative of no loss of attachment, adult of this study was to
Periodontitis, generalized severe juvenile Periodontitis, or localized juvenile Periodontitis. Whether the data were analyzed by subject or by site, no statistical differences could be found in prevalence (proportion of positive samples) among those clinical groups. The only significant difference was that localized juvenile Periodontitis subjects had higher concentrations of Aa in their Aa-positive sites than did the other clinical groups. The prevalence of Aa-positive sites in subjects without attachment loss, but who are members of families in which early onset Periodontitis is represented, was much higher than in other reports where periodontally healthy subjects were not related to early onset Periodontitis cases. This suggests that Aa may be transmitted among members of families in which one or more members has an early onset form of Periodontitis. / Periodontol 1990; 61:643-648.
Key ence;
Words:
Periodontitis/microbiology; Actinobacillus actinomycetemcomitans Iozcm-
heredity.
The term "early onset Periodontitis" (EOP) describes clinical syndromes of periodontal disease which begin in adolescence or young adulthood and progress rapidly for variable lengths of time in apparently medically healthy individuals.1'2 The group of diseases includes localized juvenile Periodontitis (LJP), involving only first molar and incisor teeth (and perhaps a very limited number of additional teeth),3 and a generalized form in which many or all teeth are affected. The generalized form has been referred to as rapidly
progressive Periodontitis,4 generalized juvenile Periodontitis,5 and generalized severe Periodontitis (SP).6 For many years clinicians and clinical investigators have
observed a familial pattern of incidence of LJP.1,7"10 Until very recently, there have been few studies which have closely examined the possibility of inheritance pattern. Several hypotheses have been proposed regarding the mode of inheritance, with autosomal recessive and X-linked dominant being the most commonly proposed mechanisms.8'11"13 'Clinical Research Center for Periodontal Diseases, Virginia Commonwealth University, Richmond, VA. Department of Periodontics, University of Alabama School of Dentistry, The University of Alabama, Birmingham, AL. Departments of Periodontics and Oral Biology, School of Dentistry, State University of New York, Buffalo, NY.
However,
no consensus on the mode of inheritance or exof environmental effects on the familial patterns in EOP has been reached.14 LJP has been related to a Gram-negative bacterial species, Actinobacillus actinomycetemcomitans (Aa), in both microbiological15-17 and immunological studies.18"23 Such studies demonstrated a much higher prevalence of either Aa or positive antibody responses to Aa in LJP subjects than in control subjects without Periodontitis. Thus, evidence exists to suggest that Aa is a periodontal pathogen in LJP. Zambón et al.15 found that only one serotype of Aa is found in a given LJP family. Of individuals within five LJP families who harbored Aa, all family members harbored the same serotype and biotype. The consistency of biotypes of Aa within families suggested that there may be transmission of the bacteria within families. The familial pattern of EOP, along with the possibility of familial transmission of Aa, led us to the present investigation. The purpose was to determine the prevalence of Aa in subgingival floras of proband cases of EOP and of their affected and non-affected family members. A proband is defined as the first individual with EOP identified in a family. The results of our investigation suggest that Aa occurs at a much higher prevalence in periodontally healthy
tent
644
J Periodontol October 1990
A. ACTINOMYCETEMCOMITANS IN FAMILIES AFFLICTED WITH PERIODONTITIS
family
members
than
the
prevalence reported by not in an EOP family.
others15'16 for healthy individuals
MATERIALS AND METHODS The human subjects for this study were identified via ascertainment of adolescents or young adults with localized juvenile Periodontitis or generalized severe Periodontitis and consequent solicitation of their family members for participation. Subjects selected for clinical examination and sampling were medically healthy as determined from histories and clinical signs. Subjects with a history of rheumatic fever, congenital heart disease, diabetes mellitus, organ transplants, and immunosuppressive or radiation therapy were excluded from the study. The clinical periodontal status of each subject was documented by a complete examination of all teeth present. The following clinical indices were measured at four sites per tooth: Plaque Index (P1I),24 Gingival Index (GI),25 probeable pocket depth (PKT), and measurement from the gingival margin to the cemento-enamel junction (CEJ), from which attachment loss (AL) was calculated.26 Data from the clinical examination were used to classify subjects into clinical groups by their attachment loss patterns. In order not to include families in which the trait might be marginal or questionable, strict criteria were established to identify a subject as an EOP proband. Once a proband was identified, all available family members were examined and classified clinically. To permit the most likely classification, somewhat less stringent criteria were applied which were still consistent with the definitions of LJP or SP. For example, some subjects presented with less attachment loss than required by our definitions of disease for probands, but nonetheless greater than could be considered "healthy." The detailed criteria used for clinical classification are given below. Probands Localized Juvenile Periodontitis (JP) required subjects of 30 years of age or less who had at least 4 mm of attachment loss (AL) on at least two permanent first molars and incisors (at least one molar must have been affected); no more than two teeth which were not first molars or incisors could be affected by 5 mm or more AL. Generalized Severe Periodontitis (SP) required subjects of 35 years of age or less who had at least 8 teeth with at least 5 mm of AL, at least 3 of which were not first molars or incisors. Members of Families of Probands No Attachment Loss (NAL) required subjects of any age who had no evidence of attachment loss (> 2 mm) at more than one site, other than that due solely to gingival recession
facial surfaces. present in variable on
Gingival inflammation degrees of severity.
may have been
Adult Periodontitis (AP) required subjects of 35 years of age or older, with evidence of Periodontitis (AL > 2 mm) at more than one site. No history of EOP or rapid loss of attachment could be detected. Localized Juvenile Periodontitis (JP) required subjects to be of the same relatively young age range and clinical patterns as for probands, except that severity of attachment loss could be less than 4 mm (but no less than 2 mm) at affected molars and incisors.
Generalized Severe Periodontitis (SP) required subjects to be of the same relatively young age and clinical patterns as for probands, except that attachment loss at multiple sites could be as little as 3 mm. However, the subject must have had attachment loss that was more excessive than would be likely for adult Periodontitis at the same age.
Microbiological Samples So that
a
large
number of sites and individuals could be
studied, a rapid means of identification of Aa in the subgin-
gival flora by indirect immunofluorescence16 was used. Plaque samples were obtained before the clinical exami-
nation at the Clinical Research Center for Periodontal Diseases, Virginia Commonwealth University. Samples from the mesials of all first molars and the distáis of all central incisors present were obtained from each subject. After superficial cleaning of the tooth surface coronal to the gingival margin, subgingival samples of the microflora were obtained by inserting three paper points to the depth of the gingival sulcus and allowing them to remain for 10 seconds. The paper points were then placed in vials containing 1 ml of Ringer's solution with 1% formalin. Vials were capped and mailed to SUNY at Buffalo where the percent of Aa in the samples was identified as previously described.16 In brief, smears of the samples were prepared on microscopic slides and stained with fluorescein-conjugated polyclonal antisera specific for the species Aa and reactive with all serotypes. The number of fluorescent cells as a percent of the total cell count was then determined. Counts were made blindly with respect to subject classification.
Statistical Analysis Evaluation of the influences of race, sex, or clinical status on prevalence of Aa was done by stepwise logistic regression. Evaluation of the influences of clinical classification on either prevalence of sites positive for Aa or on the mean levels of Aa in positive samples was done by one-way analysis of variance. The number of positive sites and mean counts in positive samples were calculated for each subject to maintain the subject as the unit of analysis.
Volume 61 Number 10
GUNSOLLEY, RANNEY, ZAMBON, BURMEISTER,
RESULTS A total of 73 subjects from 23 JP families, 12 of which had SP probands and 11 of which had LJP probands, were sampled in this study. Forty-seven additional LJP or SP subjects, for whom no family members were available for
sampling,
were also studied. These individual subjects are identified as "All" in Table 1, which provides mean age and clinical values for subjects and sampled sites. The lower section (Families) of Table 1 provides corresponding data where one or more persons in addition to the proband were sampled within a family. Consistent with previous reports,6,27'28 subjects classified as having generalized early onset disease (SP) were older on the average (34 years) than those with localized disease (LJP) (19 years). Mean age of those family members classified as having adult Periodontitis (AP) was approximately 40, while those without attachment loss (NAL) averaged 30 years of age. Mean pocket depth of the standard, preselected sample sites was greater in the two categories of EOP than for AP or NAL. The mean data indicated that, in all groups, sampled sites usually exhibited plaque and gingival inflammation. Table 2 details the age, sex, race, and detection of Aa for each subject in the 23 families included in the study. Approximately two thirds of the subjects (61%) were female, and slightly more than two-thirds (69%) were black. Prevalence (> 0%) of Aa was 49% of all SP subjects and 66% of all LJP subjects. Corresponding figures for just those EOP subjects included in family data did not significantly differ, and percent of AP and NAL subjects who were positive for Aa fell between that for SP and that for JP. Prevalence of Aa did not differ significantly among the clinical groups either by percent positive subjects (Fig. 1) or by percent positive sites (Fig. 2, left bars). Family members with no attachment loss had virtually the same percentage of positive sites (27%) as did LJP family subjects (35%). No statistically significant differences in prevalence could be found between groups according to race or sex. There was a difference between clinical groups in the amount of Aa found in positive samples. This finding is demonstrated in two different ways. First, in Figure 2 the prevalence data for Aa is displayed for different minimum
Table 1. Clinical Characteristics of
Subjects
and
Number of
All
Family (N 23) =
*
UP
=
Class*
Subjects
LJP SP
30 44
JP SP AP NAL
16 17 22
localized NAL
Periodontitis;
18
Sites 225 257
=
no
attachment loss.
SP
DISCUSSION The prevalence estimates in the present study for Aa in persons who have no periodontal attachment loss, but who are family members of a proband with EOP, are much higher than data reported previously for the prevalence of Aa in periodontally healthy individuals who are not from families in which EOP cases are represented. In fact, we found three to five times as many such persons infected. In contrast to the 56% of NAL subjects in whom we found Aa, Zambón et al.15 found only 17% of healthy subjects to have Aa and the corresponding figure in a study by Bontà et al.16 was 10%. Our data also indicate nearly twice the number of Aa-infected sites as did the study of Bontà et al. (27% vs. 15%). One explanation for the higher prevalence of Aa in EOP families as found in this study may be the transmission of the bacteria among family members. This would be consistent with the finding of Zambón et al.15 that LJP family members with Aa had the same serotype and biotype of Aa. The percent of AP subjects positive for Aa in this study agrees quite closely with that reported by Bontà et al.,16 both studies finding a higher prevalence than did Zambón et al.15 The percent of LJP subjects positive for Aa in this study was somewhat lower than in the other two studies referenced above, although the bacterial identifications were performed by the same laboratory in all three studies. A number of reports18"22 have demonstrated that LJP subjects have a higher prevalence of antibody reactivity than do other clinical groups. However, the antibody data
AL$ §
PLIt
22.0 1 32.6±1
5.1 ±.3 5.8±.2
2.8±.3 5.2+.3
1.0±.l 1.3±.l
18.8 ±1 34.2±2 27.8±3
5.9 ±.4 6.3+ .5 4.2 ±.2 2.5 ±.1
3.2±.4 6.2±.6 3.0±.4 0.1±.l
1.2±.l 1.9 + .2 1.4±.l 0.9 + .2
generalized
severe
t Data are means ± s.e.m. t Gingival margin to base of the pocket in mm. § Cemento-enamel junction to base of the pocket in
mm.
was re-
5% of the
Sites
early
645
quired subgingival flora to be considered JP had a higher prevalence level (12%) positive, subjects than did the other clinical groups (< 5%). The second manner in which this difference can be shown is in the means of the percents of Aa in positive samples (Fig. 3). The mean percent Aa in positive samples from LIP subjects was greater than for the other three clinical groups (P < 0.01). Thus, the only difference found between clinical groups was not the prevalence of Aa, but the amount of Aa found in positive samples. to be
PKTt +
38.1+2 =
percent levels of the subgingival flora. When Aa
Agef (years) +
121 99 121 120
juvenile Periodontitis;
Sampled
SCHENKEIN
onset
Periodontitis;
GIt 1.5±
1.7 +
1.6 + 1.9+
1.5 + 0.9 + AP
=
adult
646
J Periodontol October 1990
A. ACTINOMYCETEMCOMITANS IN FAMILIES AFFLICTED WITH PERIODONTITIS Table 2. Prevalence of Actinobacillus
actinomycetemcomitans (Aa) by Family +
Family
Subject*
Diagnosis
Age
Sex
ANDE7
3703 3704 1185 1190 1191 1192 1196 1198 1217 859 851
UP AP SP AP SP NAL AP AP NAL SP AP NAL SP SP LJP AP LJP AP SP AP SP AP AP LJP NAL LJP NAL NAL AP NAL AP NAL UP SP NAL LJP AP AP LJP LJP AP SP AP SP AP SP NAL SP LJP SP SP NAL SP LJP AP AP NAL AP NAL NAL NAL UP AP SP LJP LJP AP
18
F F
BARG1
BLED1
FORB2 GOOD1 HINE1 IRVI1
JOHN4 J0HN8
852 854 2871 3520 3521 807 1337 2230 3700 2380 2905 3187 574 300 1023 1182 1183
1211 1212 1213 LEWI3
MARSI
MEAD1
MILLI
ROANI ROSEI SCHOl
SMIT9
SPAUl
STIL2
TRANl WALL2
1214 1719 1721 1859 822 817 1017 1019 2902 2903
165 2858 1736 2162 1224 1738 552 553 554 555 561 562 1687 1723 1729 793 795 796 797 798 2609 2610 2379 2381 788 790
43 37 32 35 17
Racet
and
Samples/ Samplest
W
M F M
Vv
F
w
F F M
w w
w w
1/5 3/8 0/6 2/8 3/6 0/2 5/8 3/8
F
15
28
F
2/8 03 2/5
16
F F F
29
34 18 18 42 43 39 20
46 41 20
F M
1/8
F F F M M
F M F
M F
21
F F
77
F F F M
7/7
M
F F
w
M M
w w
M
F M M M
F M F F
W W W
w w
F F F F M
0.7-
1.2
0.9- 0.9 0.9- 3.7 0.9- 3.8
14.0- 14.3 3.3- 3.6 4.8- 13.9 1.0- 4.0 1.9- 3.0 1.8- 2.0 2.52.70.80.9-
2.5 2.7 4.3 3.1
40.0-40.0 0.7- 5.4 0.9- 0.9 1.4- 49.6 0.8- 12.1
0/7 2/5 2/3 0/7
1.70.8-
2.3
1.50.30.71.4-
2.7 0.7
0.8
0/5 W
W w
20
23 22 21 42
W
F F F
1.0- 4.0 0.9- 1.3 1.3- 1.3
0/6
F
49 27 34 80
W
M
20 18 14 47
55 58 27 25 53
13
M
F
1.0- 1.8 1.8- 5.0
0/8
0/8 0/8 2/4 2/8 0/7 0/8 7/8 2/3 4/4 2/5 0/8 1/4 1/5 5/8 2/2 0/8 1/8 0/6 4/8
23 24
53 32 28 17 37 37 40 28 53 35 59 39
0/7 2/8 4/6
F F M
48
3.0- 3.0 1.0- 5.8
0/8
F
F F
(Range)'
02
F
M M
44 37 35 31 51
%Aa
0/8
30 79 17 36 55 17 28
39 22
Subject
A A
0/2 0/5 0/3 1/5 4/8 1/5 4/8 0/6 1/8 0/8
1.5
5.4
2.5- 2.5
0/3
0/6 4/6
7.0
5/8 3/6
1.04.00.90.8-
0/8 0/8 5/8
1.0-23.0
6/7
0/2
9.0 7.6
2.5
Volume 61 Number 10 Table 2.
GUNSOLLEY, RANNEY, ZAMBON, BURMEISTER,
SCHENKEIN
647
(Continued) 4-
Family
Subject*
Diagnosis
Age
Sex
WILE2
2376 2455 2457 2459 3624 3625
LJP NAL
16 10 20 37 17 31
F M F F F F
WILLI 0
NAL SP LJP NAL
Racet
Samples/ Samplest
%Aa
(Range)§
0/8 2.5- 5.3 0.9- 4.5
4/8 2/8 0/7 1/8
1.0- 1.0
0/8
Each family is coded by a four letter designation followed by a number; each subject is uniquely identified a three or four digit number, black; W white; A Asian. t t Number of samples positive for Aa/Total number of samples from each subject. Range of Aa as percent of total microscopic count in those samples which contained any Aa. *
by
=
=
JP
=
SP
FAMILY LJP FAMILY SP FAMILY AP FAMILY NAL
Figure 1. Percent of subjects with Actinobacillus actinomycetemcomitans (Aa) present in at least one sample. LJP localized juvenile Periodontitis; SP generalized severe early onset Periodontitis; AP adult Periodontitis; NAL no attachment loss; Family proband and at least one additional
Criteria for Positive Samples
=
=
=
=
=
family member studied.
2. Percent of sites sampled in which Actinobacillus actinomycetemcomitans (Aa) was present. Numbers below the X-axis indicate four different criteria for considering samples positive for Aa. LJP localized juvenile Periodontitis; SP generalized severe early onset Periodontitis; adult Periodontitis; NAL AP no attachment loss.
Figure
=
=
=
do not appear to follow the high familial prevalence found in the present study for the prevalence of the bacteria. In a recent report, Gunsolley et al.23 demonstrated that NAL subjects, whether they were related to an individual with EOP or not, had a low prevalence of antibody responses to Aa (4% for white NAL subjects to 25% for black NAL subjects). This is much lower than the prevalence of Aa in NAL subjects (56%) in the present study. In contrast the prevalence of positive antibody responses in LJP subjects was high and similar to the prevalence of Aa in this study. The high prevalence of positive antibody responses suggests that LJP subjects are more prone to Aa infections which elicit antibody responses. Subjects without attachment loss may harbor the bacteria in their subgingival flora, but are less likely to have mounted an antibody response. Thus, antibody may be a better indicator of meaningful infection than is the presence of the bacteria in low numbers.
=
Black NAL and black LJP subjects have a much higher prevalence of positive antibody responses than their white counterparts.23 Coupled with the finding in the present study that there was no racial influence on the occurrence of Aa for any clinical group, the data suggest that there are different abilities in racial groups to produce antibody responses to Aa. White and black NAL and LJP subjects appear to be exposed to the bacteria to the same degree; however, black subjects have a much higher prevalence of positive antibody responses than do white subjects. In summary of the most important finding of the present study, Aa was found in more than half of the subjects with no periodontal attachment loss who are in the same family as an EOP case. This appears to be a much higher prevalence than is the case for periodontally healthy individuals who are not in such families, consistent with the possibility that Aa is transmitted among family members.
A. ACTINOMYCETEMCOMITANS IN FAMILIES AFFLICTED WITH PERIODONTITIS
648
11.
84
12.
13.
14.
15.
16.
17.
Figure 3. Percent of total microscopic count represented by Actinobaciilus actinomycetemcomitans (Aa) in samples that contained Aa (mean ± s.e.m.). LJP localized juvenile Periodontitis; SP generalized severe early no attachment loss; onset Periodontitis; AP adult Periodontitis; NAL Family proband and at least one additional family member studied. =
=
=
18.
=
=
19.
Acknowledgments
This work was supported by National Institute of Dental Research grants DE-00130, DE-05139, DE08972, and DE07479. We thank Kimberly Lake for assistance in the clinical management of subjects. Procedures for this study were approved by the institutional review committee for human subjects at Virginia Commonwealth University. REFERENCES 1. Vandesteen G, Williams B, Ebersole J, Altman L, Page R. Clinical, microbiological and immunological studies of a family with a high prevalence of early-onset Periodontitis. J Periodontol 1984; 55:159169.
3.
Ranney RR. Host factors in early onset Periodontitis. Proc Finn Dent Soc 1987; 83:131-142. Baer PN. The case of periodontosis as a clinical entity. J Periodontol
4.
Page R,
5.
6.
7.
8. 9.
10.
and prepubertal Periodontitis. J Periodontol 1985; 56:93-101. Melnick M, Shields E, Bixler E. Periodontosis: A phenotypic and genetic analysis. Oral Surg 1976; 42:32-41. Saxén L. Prevalence of juvenile Periodontitis in Finland. J Clin Periodontol 1980; 7:165-176. Long JC, Nance WE, Waring P, Burmeister JA, Ranney RR. Early onset Periodontitis: A comparison and evaluation of two proposed modes of inheritance. Genet Epidemiol 1987; 4:13-24. Boughman J, Beaty T, Yang P, Goodman S, Wooten R, Suzuki J. Problems of genetic model testing in early onset Periodontitis. / Periodontol 1988; 59:332-337. Zambón JJ, Christersson LA, Slots J. Actinobaciilus actinomycetemcomitans in human periodontal disease; prevalence in patient groups and distribution of biotypes and serotypes within families. / Periodontol 1983; 54:707-711. Bontà Y, Zambón JJ, Genco RJ, Neiders ME. Rapid identification of periodontal pathogens in subgingival plaque: Comparison of indirect immunofluorescence microscopy with bacterial culture for detection oí Actinobaciilus actinomycetemcomitans. J Dent Res 1985; 64:793-798. Christersson L, Albini , Zambón J, Wikesjö U, Genco R. Tissue localization of Actinobaciilus actinomycetemcomitans in human Periodontitis. J Periodontol 1987; 58:529-539. Listgarten MA, Lai C- , Evian CI. Comparative antibody titers to Actinobaciilus actinomycetemcomitans in juvenile Periodontitis, chronic Periodontitis and periodontally healthy subjects. J Clin Periodontol 1981; 8:155-164. Ranney RR, Yanni NR, Burmeister JA, Tew JG. Relationship between attachment loss and precipitating serum anitbody to Actinobaciilus actinomycetemcomitans in adolescents and young adults having severe periodontal destruction. J Periodontol 1982; 53:1-7. Ebersole JL, Taubman MA, Smith IJ, Genco RJ, Frey DE. Human immune responses to oral micro-organisms. I. Association of localized juvenile Periodontitis (LJP) with serum antibody responses to Actinobaciilus actinomycetemcomitans. Clin Exp Immunol 1982; 47:43-
manifesting rapidly progressive, juvenile
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J Periodontol October 1990
1971; 42:516-520. Altman L, Ebersole J,
al. Rapidly progressive Periodontitis. A distinct clinical condition. / Periodontol 1983; 54:197-209. Van Dyke TE, Horoszewicz HV, Cianciola LJ, Genco RJ. Neutrophil Chemotaxis dysfunction in human Periodontitis. Infect Immun 1980; 27:124-132. Burmeister JA, Best AM, Palcanis KG, Caine FA, Ranney RR. Localized juvenile Periodontitis and generalized severe Periodontitis: Clinical findings. / Clin Periodontol 1984; 11:181-192. Fourel J. Periodontosis: A periodontal syndrome. / Periodontol 1972; 43:240-255. Jorgenson R, Levin L, Hutcherson S, Salinas C. Periodontitis in sibs. Oral Surg 1975; 39:396^102. Hoffman I. Familial occurrence of juvenile Periodontitis with varied treatment of one of the siblings with five-year follow-up. J Periodontol 1983; 54:44-49. Spektor MD, Vandesteen GE, Page RC. Clinical studies of one family et
20.
52. 21. Vincent JW, Suzuki JB, Falkler WA Jr., Cornett WC. Reaction of human sera from juvenile Periodontitis, rapidly progressive Periodontitis and adult Periodontitis patients with selected periodontopathogens. J Periodontol 1985; 56:464-469. 22. Tew JG, Marshall DR, Moore WEC, Best AM, Palcanis KG, Ranney RR. Serum antibody reactive with predominant organisms in the subgingival flora of young adults with generalized severe Periodontitis. Infect Immun 1985; 48:303-311. 23. Gunsolley J, Tew J, Gooss C, Burmeister J, Schenkein H. Effects of race and periodontal status on antibody reactive with Actinobaciilus actinomycetemcomitans strain Y4. / Periodont Res 1988; 23:303-
307. 24. Silness J, Löe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964; 22:112-135. 25. Löe H, Silness J. Periodontal disease in pregnancy. I Prevalence and severity. Acta Odontol Scand 1963; 21:533-551. 26. Glavind L, Löe H. Errors in the clinical assessment of periodontal destruction. J Periodont Res 1967; 2:179-184. 27. H0rmand J, Frandsen A. Juvenile Periodontitis. Localization of bone loss in relation to age, sex, and teeth. / Clin Periodontol 1979; 6:407-
416. 28. Saxén L, Murtomaa H. Age-related expression of juvenile titis. J Clin Periodontol 1985; 12:21-26.
Periodon-
Send reprint requests to: Dr. John C. Gunsolley, Virginia Commonwealth University, MCV Station, Box 566, Richmond, VA 23298-0001. Accepted for publication April 16, 1990.
The purpose
Ranney,f Joseph J. Zambón,# John A. Burmeister,
*
determine the prevalence of Actinobacillus actinoin individuals from families where at least one individual has an mycetemcomitans (Aa) form of onset Periodontitis. Twenty-three families with 73 subjects were evaluated early in this study. Forty-seven early onset Periodontitis subjects outside the 23 families were also studied. Prevalence of detection of Aa in family members ranged from 49% to 66% among groups constituted by clinical findings indicative of no loss of attachment, adult of this study was to
Periodontitis, generalized severe juvenile Periodontitis, or localized juvenile Periodontitis. Whether the data were analyzed by subject or by site, no statistical differences could be found in prevalence (proportion of positive samples) among those clinical groups. The only significant difference was that localized juvenile Periodontitis subjects had higher concentrations of Aa in their Aa-positive sites than did the other clinical groups. The prevalence of Aa-positive sites in subjects without attachment loss, but who are members of families in which early onset Periodontitis is represented, was much higher than in other reports where periodontally healthy subjects were not related to early onset Periodontitis cases. This suggests that Aa may be transmitted among members of families in which one or more members has an early onset form of Periodontitis. / Periodontol 1990; 61:643-648.
Key ence;
Words:
Periodontitis/microbiology; Actinobacillus actinomycetemcomitans Iozcm-
heredity.
The term "early onset Periodontitis" (EOP) describes clinical syndromes of periodontal disease which begin in adolescence or young adulthood and progress rapidly for variable lengths of time in apparently medically healthy individuals.1'2 The group of diseases includes localized juvenile Periodontitis (LJP), involving only first molar and incisor teeth (and perhaps a very limited number of additional teeth),3 and a generalized form in which many or all teeth are affected. The generalized form has been referred to as rapidly
progressive Periodontitis,4 generalized juvenile Periodontitis,5 and generalized severe Periodontitis (SP).6 For many years clinicians and clinical investigators have
observed a familial pattern of incidence of LJP.1,7"10 Until very recently, there have been few studies which have closely examined the possibility of inheritance pattern. Several hypotheses have been proposed regarding the mode of inheritance, with autosomal recessive and X-linked dominant being the most commonly proposed mechanisms.8'11"13 'Clinical Research Center for Periodontal Diseases, Virginia Commonwealth University, Richmond, VA. Department of Periodontics, University of Alabama School of Dentistry, The University of Alabama, Birmingham, AL. Departments of Periodontics and Oral Biology, School of Dentistry, State University of New York, Buffalo, NY.
However,
no consensus on the mode of inheritance or exof environmental effects on the familial patterns in EOP has been reached.14 LJP has been related to a Gram-negative bacterial species, Actinobacillus actinomycetemcomitans (Aa), in both microbiological15-17 and immunological studies.18"23 Such studies demonstrated a much higher prevalence of either Aa or positive antibody responses to Aa in LJP subjects than in control subjects without Periodontitis. Thus, evidence exists to suggest that Aa is a periodontal pathogen in LJP. Zambón et al.15 found that only one serotype of Aa is found in a given LJP family. Of individuals within five LJP families who harbored Aa, all family members harbored the same serotype and biotype. The consistency of biotypes of Aa within families suggested that there may be transmission of the bacteria within families. The familial pattern of EOP, along with the possibility of familial transmission of Aa, led us to the present investigation. The purpose was to determine the prevalence of Aa in subgingival floras of proband cases of EOP and of their affected and non-affected family members. A proband is defined as the first individual with EOP identified in a family. The results of our investigation suggest that Aa occurs at a much higher prevalence in periodontally healthy
tent
644
J Periodontol October 1990
A. ACTINOMYCETEMCOMITANS IN FAMILIES AFFLICTED WITH PERIODONTITIS
family
members
than
the
prevalence reported by not in an EOP family.
others15'16 for healthy individuals
MATERIALS AND METHODS The human subjects for this study were identified via ascertainment of adolescents or young adults with localized juvenile Periodontitis or generalized severe Periodontitis and consequent solicitation of their family members for participation. Subjects selected for clinical examination and sampling were medically healthy as determined from histories and clinical signs. Subjects with a history of rheumatic fever, congenital heart disease, diabetes mellitus, organ transplants, and immunosuppressive or radiation therapy were excluded from the study. The clinical periodontal status of each subject was documented by a complete examination of all teeth present. The following clinical indices were measured at four sites per tooth: Plaque Index (P1I),24 Gingival Index (GI),25 probeable pocket depth (PKT), and measurement from the gingival margin to the cemento-enamel junction (CEJ), from which attachment loss (AL) was calculated.26 Data from the clinical examination were used to classify subjects into clinical groups by their attachment loss patterns. In order not to include families in which the trait might be marginal or questionable, strict criteria were established to identify a subject as an EOP proband. Once a proband was identified, all available family members were examined and classified clinically. To permit the most likely classification, somewhat less stringent criteria were applied which were still consistent with the definitions of LJP or SP. For example, some subjects presented with less attachment loss than required by our definitions of disease for probands, but nonetheless greater than could be considered "healthy." The detailed criteria used for clinical classification are given below. Probands Localized Juvenile Periodontitis (JP) required subjects of 30 years of age or less who had at least 4 mm of attachment loss (AL) on at least two permanent first molars and incisors (at least one molar must have been affected); no more than two teeth which were not first molars or incisors could be affected by 5 mm or more AL. Generalized Severe Periodontitis (SP) required subjects of 35 years of age or less who had at least 8 teeth with at least 5 mm of AL, at least 3 of which were not first molars or incisors. Members of Families of Probands No Attachment Loss (NAL) required subjects of any age who had no evidence of attachment loss (> 2 mm) at more than one site, other than that due solely to gingival recession
facial surfaces. present in variable on
Gingival inflammation degrees of severity.
may have been
Adult Periodontitis (AP) required subjects of 35 years of age or older, with evidence of Periodontitis (AL > 2 mm) at more than one site. No history of EOP or rapid loss of attachment could be detected. Localized Juvenile Periodontitis (JP) required subjects to be of the same relatively young age range and clinical patterns as for probands, except that severity of attachment loss could be less than 4 mm (but no less than 2 mm) at affected molars and incisors.
Generalized Severe Periodontitis (SP) required subjects to be of the same relatively young age and clinical patterns as for probands, except that attachment loss at multiple sites could be as little as 3 mm. However, the subject must have had attachment loss that was more excessive than would be likely for adult Periodontitis at the same age.
Microbiological Samples So that
a
large
number of sites and individuals could be
studied, a rapid means of identification of Aa in the subgin-
gival flora by indirect immunofluorescence16 was used. Plaque samples were obtained before the clinical exami-
nation at the Clinical Research Center for Periodontal Diseases, Virginia Commonwealth University. Samples from the mesials of all first molars and the distáis of all central incisors present were obtained from each subject. After superficial cleaning of the tooth surface coronal to the gingival margin, subgingival samples of the microflora were obtained by inserting three paper points to the depth of the gingival sulcus and allowing them to remain for 10 seconds. The paper points were then placed in vials containing 1 ml of Ringer's solution with 1% formalin. Vials were capped and mailed to SUNY at Buffalo where the percent of Aa in the samples was identified as previously described.16 In brief, smears of the samples were prepared on microscopic slides and stained with fluorescein-conjugated polyclonal antisera specific for the species Aa and reactive with all serotypes. The number of fluorescent cells as a percent of the total cell count was then determined. Counts were made blindly with respect to subject classification.
Statistical Analysis Evaluation of the influences of race, sex, or clinical status on prevalence of Aa was done by stepwise logistic regression. Evaluation of the influences of clinical classification on either prevalence of sites positive for Aa or on the mean levels of Aa in positive samples was done by one-way analysis of variance. The number of positive sites and mean counts in positive samples were calculated for each subject to maintain the subject as the unit of analysis.
Volume 61 Number 10
GUNSOLLEY, RANNEY, ZAMBON, BURMEISTER,
RESULTS A total of 73 subjects from 23 JP families, 12 of which had SP probands and 11 of which had LJP probands, were sampled in this study. Forty-seven additional LJP or SP subjects, for whom no family members were available for
sampling,
were also studied. These individual subjects are identified as "All" in Table 1, which provides mean age and clinical values for subjects and sampled sites. The lower section (Families) of Table 1 provides corresponding data where one or more persons in addition to the proband were sampled within a family. Consistent with previous reports,6,27'28 subjects classified as having generalized early onset disease (SP) were older on the average (34 years) than those with localized disease (LJP) (19 years). Mean age of those family members classified as having adult Periodontitis (AP) was approximately 40, while those without attachment loss (NAL) averaged 30 years of age. Mean pocket depth of the standard, preselected sample sites was greater in the two categories of EOP than for AP or NAL. The mean data indicated that, in all groups, sampled sites usually exhibited plaque and gingival inflammation. Table 2 details the age, sex, race, and detection of Aa for each subject in the 23 families included in the study. Approximately two thirds of the subjects (61%) were female, and slightly more than two-thirds (69%) were black. Prevalence (> 0%) of Aa was 49% of all SP subjects and 66% of all LJP subjects. Corresponding figures for just those EOP subjects included in family data did not significantly differ, and percent of AP and NAL subjects who were positive for Aa fell between that for SP and that for JP. Prevalence of Aa did not differ significantly among the clinical groups either by percent positive subjects (Fig. 1) or by percent positive sites (Fig. 2, left bars). Family members with no attachment loss had virtually the same percentage of positive sites (27%) as did LJP family subjects (35%). No statistically significant differences in prevalence could be found between groups according to race or sex. There was a difference between clinical groups in the amount of Aa found in positive samples. This finding is demonstrated in two different ways. First, in Figure 2 the prevalence data for Aa is displayed for different minimum
Table 1. Clinical Characteristics of
Subjects
and
Number of
All
Family (N 23) =
*
UP
=
Class*
Subjects
LJP SP
30 44
JP SP AP NAL
16 17 22
localized NAL
Periodontitis;
18
Sites 225 257
=
no
attachment loss.
SP
DISCUSSION The prevalence estimates in the present study for Aa in persons who have no periodontal attachment loss, but who are family members of a proband with EOP, are much higher than data reported previously for the prevalence of Aa in periodontally healthy individuals who are not from families in which EOP cases are represented. In fact, we found three to five times as many such persons infected. In contrast to the 56% of NAL subjects in whom we found Aa, Zambón et al.15 found only 17% of healthy subjects to have Aa and the corresponding figure in a study by Bontà et al.16 was 10%. Our data also indicate nearly twice the number of Aa-infected sites as did the study of Bontà et al. (27% vs. 15%). One explanation for the higher prevalence of Aa in EOP families as found in this study may be the transmission of the bacteria among family members. This would be consistent with the finding of Zambón et al.15 that LJP family members with Aa had the same serotype and biotype of Aa. The percent of AP subjects positive for Aa in this study agrees quite closely with that reported by Bontà et al.,16 both studies finding a higher prevalence than did Zambón et al.15 The percent of LJP subjects positive for Aa in this study was somewhat lower than in the other two studies referenced above, although the bacterial identifications were performed by the same laboratory in all three studies. A number of reports18"22 have demonstrated that LJP subjects have a higher prevalence of antibody reactivity than do other clinical groups. However, the antibody data
AL$ §
PLIt
22.0 1 32.6±1
5.1 ±.3 5.8±.2
2.8±.3 5.2+.3
1.0±.l 1.3±.l
18.8 ±1 34.2±2 27.8±3
5.9 ±.4 6.3+ .5 4.2 ±.2 2.5 ±.1
3.2±.4 6.2±.6 3.0±.4 0.1±.l
1.2±.l 1.9 + .2 1.4±.l 0.9 + .2
generalized
severe
t Data are means ± s.e.m. t Gingival margin to base of the pocket in mm. § Cemento-enamel junction to base of the pocket in
mm.
was re-
5% of the
Sites
early
645
quired subgingival flora to be considered JP had a higher prevalence level (12%) positive, subjects than did the other clinical groups (< 5%). The second manner in which this difference can be shown is in the means of the percents of Aa in positive samples (Fig. 3). The mean percent Aa in positive samples from LIP subjects was greater than for the other three clinical groups (P < 0.01). Thus, the only difference found between clinical groups was not the prevalence of Aa, but the amount of Aa found in positive samples. to be
PKTt +
38.1+2 =
percent levels of the subgingival flora. When Aa
Agef (years) +
121 99 121 120
juvenile Periodontitis;
Sampled
SCHENKEIN
onset
Periodontitis;
GIt 1.5±
1.7 +
1.6 + 1.9+
1.5 + 0.9 + AP
=
adult
646
J Periodontol October 1990
A. ACTINOMYCETEMCOMITANS IN FAMILIES AFFLICTED WITH PERIODONTITIS Table 2. Prevalence of Actinobacillus
actinomycetemcomitans (Aa) by Family +
Family
Subject*
Diagnosis
Age
Sex
ANDE7
3703 3704 1185 1190 1191 1192 1196 1198 1217 859 851
UP AP SP AP SP NAL AP AP NAL SP AP NAL SP SP LJP AP LJP AP SP AP SP AP AP LJP NAL LJP NAL NAL AP NAL AP NAL UP SP NAL LJP AP AP LJP LJP AP SP AP SP AP SP NAL SP LJP SP SP NAL SP LJP AP AP NAL AP NAL NAL NAL UP AP SP LJP LJP AP
18
F F
BARG1
BLED1
FORB2 GOOD1 HINE1 IRVI1
JOHN4 J0HN8
852 854 2871 3520 3521 807 1337 2230 3700 2380 2905 3187 574 300 1023 1182 1183
1211 1212 1213 LEWI3
MARSI
MEAD1
MILLI
ROANI ROSEI SCHOl
SMIT9
SPAUl
STIL2
TRANl WALL2
1214 1719 1721 1859 822 817 1017 1019 2902 2903
165 2858 1736 2162 1224 1738 552 553 554 555 561 562 1687 1723 1729 793 795 796 797 798 2609 2610 2379 2381 788 790
43 37 32 35 17
Racet
and
Samples/ Samplest
W
M F M
Vv
F
w
F F M
w w
w w
1/5 3/8 0/6 2/8 3/6 0/2 5/8 3/8
F
15
28
F
2/8 03 2/5
16
F F F
29
34 18 18 42 43 39 20
46 41 20
F M
1/8
F F F M M
F M F
M F
21
F F
77
F F F M
7/7
M
F F
w
M M
w w
M
F M M M
F M F F
W W W
w w
F F F F M
0.7-
1.2
0.9- 0.9 0.9- 3.7 0.9- 3.8
14.0- 14.3 3.3- 3.6 4.8- 13.9 1.0- 4.0 1.9- 3.0 1.8- 2.0 2.52.70.80.9-
2.5 2.7 4.3 3.1
40.0-40.0 0.7- 5.4 0.9- 0.9 1.4- 49.6 0.8- 12.1
0/7 2/5 2/3 0/7
1.70.8-
2.3
1.50.30.71.4-
2.7 0.7
0.8
0/5 W
W w
20
23 22 21 42
W
F F F
1.0- 4.0 0.9- 1.3 1.3- 1.3
0/6
F
49 27 34 80
W
M
20 18 14 47
55 58 27 25 53
13
M
F
1.0- 1.8 1.8- 5.0
0/8
0/8 0/8 2/4 2/8 0/7 0/8 7/8 2/3 4/4 2/5 0/8 1/4 1/5 5/8 2/2 0/8 1/8 0/6 4/8
23 24
53 32 28 17 37 37 40 28 53 35 59 39
0/7 2/8 4/6
F F M
48
3.0- 3.0 1.0- 5.8
0/8
F
F F
(Range)'
02
F
M M
44 37 35 31 51
%Aa
0/8
30 79 17 36 55 17 28
39 22
Subject
A A
0/2 0/5 0/3 1/5 4/8 1/5 4/8 0/6 1/8 0/8
1.5
5.4
2.5- 2.5
0/3
0/6 4/6
7.0
5/8 3/6
1.04.00.90.8-
0/8 0/8 5/8
1.0-23.0
6/7
0/2
9.0 7.6
2.5
Volume 61 Number 10 Table 2.
GUNSOLLEY, RANNEY, ZAMBON, BURMEISTER,
SCHENKEIN
647
(Continued) 4-
Family
Subject*
Diagnosis
Age
Sex
WILE2
2376 2455 2457 2459 3624 3625
LJP NAL
16 10 20 37 17 31
F M F F F F
WILLI 0
NAL SP LJP NAL
Racet
Samples/ Samplest
%Aa
(Range)§
0/8 2.5- 5.3 0.9- 4.5
4/8 2/8 0/7 1/8
1.0- 1.0
0/8
Each family is coded by a four letter designation followed by a number; each subject is uniquely identified a three or four digit number, black; W white; A Asian. t t Number of samples positive for Aa/Total number of samples from each subject. Range of Aa as percent of total microscopic count in those samples which contained any Aa. *
by
=
=
JP
=
SP
FAMILY LJP FAMILY SP FAMILY AP FAMILY NAL
Figure 1. Percent of subjects with Actinobacillus actinomycetemcomitans (Aa) present in at least one sample. LJP localized juvenile Periodontitis; SP generalized severe early onset Periodontitis; AP adult Periodontitis; NAL no attachment loss; Family proband and at least one additional
Criteria for Positive Samples
=
=
=
=
=
family member studied.
2. Percent of sites sampled in which Actinobacillus actinomycetemcomitans (Aa) was present. Numbers below the X-axis indicate four different criteria for considering samples positive for Aa. LJP localized juvenile Periodontitis; SP generalized severe early onset Periodontitis; adult Periodontitis; NAL AP no attachment loss.
Figure
=
=
=
do not appear to follow the high familial prevalence found in the present study for the prevalence of the bacteria. In a recent report, Gunsolley et al.23 demonstrated that NAL subjects, whether they were related to an individual with EOP or not, had a low prevalence of antibody responses to Aa (4% for white NAL subjects to 25% for black NAL subjects). This is much lower than the prevalence of Aa in NAL subjects (56%) in the present study. In contrast the prevalence of positive antibody responses in LJP subjects was high and similar to the prevalence of Aa in this study. The high prevalence of positive antibody responses suggests that LJP subjects are more prone to Aa infections which elicit antibody responses. Subjects without attachment loss may harbor the bacteria in their subgingival flora, but are less likely to have mounted an antibody response. Thus, antibody may be a better indicator of meaningful infection than is the presence of the bacteria in low numbers.
=
Black NAL and black LJP subjects have a much higher prevalence of positive antibody responses than their white counterparts.23 Coupled with the finding in the present study that there was no racial influence on the occurrence of Aa for any clinical group, the data suggest that there are different abilities in racial groups to produce antibody responses to Aa. White and black NAL and LJP subjects appear to be exposed to the bacteria to the same degree; however, black subjects have a much higher prevalence of positive antibody responses than do white subjects. In summary of the most important finding of the present study, Aa was found in more than half of the subjects with no periodontal attachment loss who are in the same family as an EOP case. This appears to be a much higher prevalence than is the case for periodontally healthy individuals who are not in such families, consistent with the possibility that Aa is transmitted among family members.
A. ACTINOMYCETEMCOMITANS IN FAMILIES AFFLICTED WITH PERIODONTITIS
648
11.
84
12.
13.
14.
15.
16.
17.
Figure 3. Percent of total microscopic count represented by Actinobaciilus actinomycetemcomitans (Aa) in samples that contained Aa (mean ± s.e.m.). LJP localized juvenile Periodontitis; SP generalized severe early no attachment loss; onset Periodontitis; AP adult Periodontitis; NAL Family proband and at least one additional family member studied. =
=
=
18.
=
=
19.
Acknowledgments
This work was supported by National Institute of Dental Research grants DE-00130, DE-05139, DE08972, and DE07479. We thank Kimberly Lake for assistance in the clinical management of subjects. Procedures for this study were approved by the institutional review committee for human subjects at Virginia Commonwealth University. REFERENCES 1. Vandesteen G, Williams B, Ebersole J, Altman L, Page R. Clinical, microbiological and immunological studies of a family with a high prevalence of early-onset Periodontitis. J Periodontol 1984; 55:159169.
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J Periodontol October 1990
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Periodon-
Send reprint requests to: Dr. John C. Gunsolley, Virginia Commonwealth University, MCV Station, Box 566, Richmond, VA 23298-0001. Accepted for publication April 16, 1990.
