Carlos E. POMES2
Walter A. BRETZ1
Alfonso de LEON2
Eros S. CHAVES3
1University of Pittsburgh, School of Dental Medicine,
Pittsburgh, PA, USA
2Universidad San Carlos de Guatemala, Guatemala City, Guatemala
3Hilltop Research, West Palm Beach, FL, USA
Braz Dent J (2000) 11(1): 49-57 ISSN 0103-6440
Introduction | Material and Methods | Results | Discussion | Resumo | References
A random sample of sixty-two 11-15-year-old adolescents from 17 different locations in Guatemala were selected for this study. Pocket depth, Plaque Index, and bleeding upon probing were recorded from 6 randomly selected sites in each subject (a total of 372 sites). Subgingival plaque samples were subsequently collected from these sites and processed by several assays. For cost reasons, in each pair of sites different assays were performed as follows: sites #1, #2 - BANA test for T. denticola, P. gingivalis, B. forsythus and screening of plaque samples with polyclonal antibodies (ELISA system) for A. actinomycetemcomitans; sites #3, #4 - detection of yeasts by SAB agar; sites #5, #6 - detection of Entamoeba gingivalis by the Heidenhain iron hematoxylin modified technique. A total of 66% of the children had at least one site that bled upon probing, 42% exhibited at least one site with pocket depth >3 mm, and 79% exhibited a high Plaque Index, with the percent of sites affected being 30%, 12% and 41%, respectively. In sites #1, #2 (N = 124), the BANA test assay and A. actinomycetemcomitans tested positive in 77% and 47% of the children accounting for 59% and 31% of the sites, respectively. In sites #3, #4 (N = 124), yeasts were detected in 43% of the children and 29% of the sites. In sites #5, #6 (N = 124), Entamoeba gingivalis was detected in 21% of the children and in 11% of the sites. The risk for severe gingival inflammation and/or increased probing depth was 1.5 and 5.2 times higher if a positive BANA test or A. actinomycetemcomitans test was found in a particular site. No associations could be found for yeasts and Entamoeba gingivalis.
Key Words: risk indicators, periodontal diseases, adolescents, BANA test, ELISA.
The association of Actinobacillus actinomycetemcomitans and destructive periodontal diseases in adolescents and young adults has been reported (Zambon, 1985). Periodontal diseases affecting adults are frequently associated with periodontal pathogens such as Treponema denticola, Porphyromonas gingivalis and Bacteroides forsythus (Loesche et al., 1985; Moore, 1987; Dzink et al., 1988). Recently, an enzyme assay that detects T. denticola, P. gingivalis or B. forsythus (BANA testTM) was found to be positive for these organisms at a moderate frequency in plaque samples collected from the gingival crevice of children and adolescents (Watson et al., 1991).
In most of the rural areas of Guatemala, children and adolescents are undernourished and frequently have high levels of undisturbed dental plaque aggregates (INCAP, 1989). Studies of 12-16-year-old Guatemalan adolescents have reported that gingivitis affects the vast majority of individuals, and that the prevalence of irreversible forms of periodontal disease can be observed in 12-36% of the individuals (Moll, 1984; Sandoval-Godoy, 1984; Pomes et al., 1985).
Because little is known about the prevalence of periodontopathic bacteria as well as yeasts and parasites in the gingival domain, the purpose of this study was to determine if the subgingival microflora of Guatemalan adolescents would test positive for the BANA test, A. actinomycetemcomitans, yeasts and parasites, and if so, whether associations between these organisms and the occurrence of periodontal diseases could be observed.
Material and Methods
The sample consisted of 62 Guatemalan (33 males and 29 females) 11-15-year-old adolescents (mean age 12.9 years) randomly selected from public schools of 17 villages located in the southern and western part of the country. Subjects had no history of antibiotic therapy in the previous 3 months and no systemic illness was diagnosed at the time of the study.
Periodontal site selection and clinical parameters
Periodontal clinical examination was performed by a calibrated examiner on 6 randomly selected sites from each subject (a total of 372 sites). Sites were numbered from #1 to #6 following the sequence of their random selection beginning in the right upper quadrant. Twenty-two percent of the examined sites were in the molar region, 29% in the premolar region and 49% in the anterior region. Examination procedures were carried out with the aid of dental chairs, artificial lights, dental mirrors, subgingival curettes and periodontal probes at health centers in each of the villages. Clinical measurements performed on each of the six selected sites included the presence or absence of plaque as determined by the Plaque Index (Silness and Löe, 1964), the clinical probing depth of the gingival sulcus or pocket, and bleeding upon probing. Efforts were made to characterize the flora inhabiting the periodontal pocket and subsequently to relate it with increased probing depth. Following the measurement of clinical parameters, the supragingival plaque was removed with a curette and discarded, and subgingival plaque samples were then taken from the most apical portion of the gingival sulcus or pocket.
Plaque sampling and microbial assays
Subgingival plaque samples were processed by various assays. Due to cost restraints, different assays were performed on pairs of sites. In sites #1 and #2, the BANA test for the detection of T. denticola, P. gingivalis, and B. forsythus, and the ELISA system for the detection of A. actinomycetemcomitans (Y4) utilizing species-specific polyclonal antibodies were performed using methods described elsewhere (Bretz et al., 1990; Loesche et al., 1990). It should be noted that we obtained the information both on the BANA test and on the presence of A. actinomycetemcomitans simultaneously in the same plaque sample. Thus, after reading the results of the BANA test, the plaque sample on the BANA test card was stained with a polyclonal antibody for A. actinomycetemcomitans. In sites #3 and #4, SAB medium (Saboraud dextrose agar) was used to detect the presence or absence of yeasts in plaque samples. In sites #5 and #6, the plaque smears were fixed with Turdyev fixative solution and then stained with the Heidenhain iron hematoxylin modified technique (Lynch et al., 1969). The samples were examined under phase and light microscopes for Entamoeba gingivalis.
The clinical and microbiological data were dichotomized, as follows: a) Plaque Index scores (0-1, 2-3); b) pocket depth (£3 mm, >3 mm); c) bleeding upon probing (no, yes); d) BANA test and ELISA for A. actinomycetemcomitans (negative, positive); e) yeasts and E. gingivalis (negative, positive). The data were treated similar to those of a case-control study, with sites exhibiting increased probing depth (>3 mm) being classified as cases. Controls were sites considered clinically healthy with probing depth £3 mm and no bleeding upon probing. This permitted the use of epidemiologic measures of risk (odds ratio and 95% confidence intervals). Some of the clinical and microbiological parameters were treated as possible confounders, and their effect was controlled by stratification. For the stratified analysis, the Mantel-Haenszel procedure was used for calculation of summary odds ratios over strata, and the significance of the odds ratio was tested by the Mantel-Haenszel chi-square test. The binomial model used in this study did not take into account the fact that sites within an individual's mouth may be correlated. Analysis with a correlated binomial model indicated that there was a low degree of correlation among the sites sampled, and consequently the results of this study generated by ordinary binomial models were unaffected.
The distribution of the clinical parameters by subjects and by sites is shown in Table 1. Forty-two percent of the subjects presented at least one pocket depth >3 mm, accounting for a small fraction of the sites (12%). The presence of visible accumulations of plaque and/or calculus were frequently observed (79%), accounting for 41% of the sites. The frequency of bleeding was high (66%), accounting for 30% of the sites. The prevalence of the selected microbiological parameters is shown in Table 2. Seventy-seven percent, 47%, 43% and 21% of the subjects, respectively, tested positive for the BANA test, A. actinomycetemco mitans, yeasts and E. gingivalis, accounting for 59%, 31%, 29% and 11% of the sites, respectively.
The observation of increased probing depth with the presence of organisms screened in this study and the presence of plaque and bleeding is summarized in Table 3. It can be observed that the detection of A. actinomycetemcomitans significantly increased the likelihood of having a deep periodontal pocket at a particular site (OR = 5.16). The presence of a positive BANA test increased the likelihood of a site having increased probing depth (OR = 1.46), although the 95% confidence interval included 1.0. No positive associations were found for the presence of yeasts and E. gingivalis. The presence of large or at least visible amounts of plaque and of bleeding increased the likelihood of a site having increased probing depth.
The effects of A. actinomycetemcomitans on increased probing depth were adjusted for the presence of plaque, bleeding, or both, and for the BANA test (Table 4). The relationship between A. actinomycetemcomitans and increased probing depth was significantly confounded by the presence of bleeding and plaque. The presence of both plaque and bleeding reduced the effects of A. actinomycetemcomitans on increased probing depth, and these effects were of borderline significance (confidence interval included 1.0). Lastly, the presence of a positive BANA test sharply decreased the effects of a positive test for A. actinomycetemcomitans on increased probing depth, although the confidence interval included 1.0.
In this study, 42% of the subjects presented with at least one of the examined sites with probing depth >3 mm, accounting for 12% of the sites. A previous study on the prevalence of periodontal diseases in 15-year-old Guatemalan adolescents of two rural communities reported that 12 to 36% of these adolescents presented with at least one site with some form of destructive periodontitis (Moll, 1984; Sandoval-Godoy, 1984). In addition, a study of 12-13-year-old adolescents from a village in Guatemala showed that 49% of the subjects presented with a Periodontal Index of 6 in at least one site, suggesting the presence of pockets (Moll, 1984). This prevalence is considerably higher than reported for Swedish and Finish adolescents (Kallestal et al., 1990; Asikainen et al., 1991). In the Swedish study, 4% of the subjects presented with one or more sites with probing depth >3 mm (Kallestal et al., 1990) whereas in the Finish adolescents no site exhibited a probing depth exceeding 3 mm (Asikainen et al., 1991). This suggests that the prevalence of periodontal destruction and/or severe gingival inflammation is more prevalent in Guatemalan adolescents than in Scandinavian adolescents.
However, the number of sites examined in an individual and the methods and criteria used to assess periodontal destruction in these studies varied considerably, making comparisons of the prevalence of periodontal diseases difficult.
The Guatemalan adolescents in the present study had a high prevalence of BANA test-positive subgingival plaque samples (59% of the 124 plaque samples were positive). Studies in pre-school US children have indicated that 21% of 620 plaque samples tested positive for the BANA test (Watson et al., 1991). These data could suggest that the frequency of colonization by BANA-positive species increases with age, but could also suggest that the Guatemalans are more highly colonized. Malnutrition is highly prevalent among Guatemalan children (Sandoval-Godoy, 1984). Since malnutrition is a host debilitating condition, a proliferation of oral organisms might be expected. Indeed, comparison of the flora of well-nourished and mal-nourished Nigerian children has shown clear differences in terms of the proportions and types of organisms found in their gingival domain (Sawyer et al., 1986). Eighty-eight percent of the plaque samples taken from malnourished Nigerian children presented with high proportions of spirochetes, whereas spirochetes were not detected in any of the samples of the well-nourished children. Similarly, black-pigmented anaerobic rods were often isolated from malnourished children, and were not detected in well-nourished children. The BANA test detects indirectly the presence of spirochetes and Porphyromonas gingivalis. Given the level of malnutrition in Guatemalan children that may affect the periodontal flora, as observed in studies of malnourished children in Nigeria, the high prevalence of BANA test-positive plaque samples was likely to be observed.
A. actinomycetemcomitans was detected in 47% of the subjects, and in 31% of 124 plaque samples. This prevalence is higher than previously reported prevalences of A. actinomycetemcomitans using conventional culturing techniques (Zambon, 1985). It is possible that the species-specific immunological reagents used in this study are more sensitive than culture methods in detecting the presence of A. actinomycetemcomitans. Several studies have shown that culture methods are not as effective as DNA probes, immunological reagents and enzymatic assays in detecting the presence of suspected periodontal pathogens, including A. actinomycetemcomitans (Loesche et al., 1992).
The levels of oral hygiene in the population studied are poor as are the sanitary conditions in which they live. It was of interest to study the occurrence of parasites and yeasts in the periodontal flora of Guatemalan adolescents. A previous study in Guatemala (De Leon and Rosal, 1988) showed that 15% of young adults had E. gingivalis detected in subgingival plaque samples, a value which is in agreement with the 21% prevalence found in this study. Studies in young French adults revealed that E. gingivalis was detected in 50% of the subjects (Feki and Molet, 1990). The presence of yeasts has not been determined in studies involving adolescents. One study from Chile (Gonzales et al., 1987) involving young adults with juvenile periodontitis reported that yeasts could be found in the connective tissue of these patients using scanning electron microscopy. In the present study yeasts were detected in 43% of the subjects accounting for 29% of the sites. The importance of yeasts and parasites in the periodontal domain remains to be studied as little is known about the interaction of these organisms with the host.
We investigated the association of clinical and microbiological parameters with the prevalence of increased probing depth (>3 mm). A. actinomycetemcomitans best explained the site-specific presence of increased probing depth, whereas none of the other monitored bacteria variables were significantly associated with an increase in probing depth (Table 3). The relationship of A. actinomycetemcomitans with increased probing depth was reduced by the presence of bleeding. Conversely, the presence of large amounts of plaque enhanced the A. actinomycetemcomitans effects on the likelihood of a site having increased probing depth. The combined effect of A. actinomycetemcomitans, bleeding and plaque best explained the presence of a site having increased probing depth, but this association was of borderline significance. The strength of the association of A. actinomycetemcomitans with increased probing depth was sharply reduced by the presence of a positive BANA test suggesting that a combination of tests should be used in order to better explain the increased likelihood of a site having increased probing depth.
The present study was an attempt to monitor in the field for the presence of putative periodontal pathogens using simple procedures. The subjects studied were of a low socio-economic status from different parts of Guatemala, but were not representative of the entire Guatemalan adolescent population. The results show that periodontal pathogens can be detected by simple methods in the field, and that A. actinomycetemcomitans could be best associated with severe gingival inflammation and/or increased probing depth. Results of these studies may be important to point out the directions of preventive community-based programs in low-industrialized countries.
Pomes CE, Bretz WA, Leon A, Aguirre R, Milian E, Chaves ES: Indicadores de risco para as doenças periodontais em adolescentes guatemaltecos. Braz Dent J 11(1): 49-57, 2000.
Uma amostra selecionada ao acaso de 62 adolescentes na faixa etária de 11 a 15 anos, de 17 localidades diferentes na Guatemala, compos o grupo a ser estudado. Profundidade de bolsa, Índice de Placa, e sangramento à sondagem foram computados de 6 sítios periodontais escolhidos ao acaso de cada participante (total de 372 sítios). Amostras de placa subgengival foram obtidas destes mesmos sítios. Em cada par de sítios, testes microbiológicos distintos foram processados, à saber: sítios 1 e 2 - teste BANA para T. denticola, P. gingivalis, e B. forsythus, e anticorpos policlonais para A. actinomycetemcomitans; sítios 3 e 4 - detecção de fungos no agar SAB; sítios 5 e 6 - detecção de Entamoeba gingivalis pela técnica de Heidenhain. Um total de 66% dos participantes evidenciaram ao menos um sítio com sangramento à sondagem, 42% exibiram no mínimo um sítio com profundidade de bolsa >3mm, e 79% exibiram um alto Índice de Placa. O percentual correspondente de sítios foi de 30%, 12% e 41%. Nos sítios 1 e 2 (N =124), 77% e 47% dos participantes testaram positivo para o teste BANA e para A. actinomycetemcomitans, respectivamente, totalizando 59% e 31% dos sítios. Nos sítios 3 e 4 (N = 124), fungos foram detectados em 43% dos participantes e 39% dos sítios. Nos sítios 5 e 6 (N = 124), E. gingivalis foi detectada em 21% dos participantes e em 11% dos sítios. O risco para inflamação gengival severa e/ou profundidade de bolsa aumentada estava 1,5 e 5,2 vezes aumentado se um resultado positivo para o teste de BANA ou para A. actinomycetemcomitans foi detectado num determinado sítio. Nenhuma associação foi verificada para fungos e para E. gingivalis.
Unitermos: indicadores de risco, doenças periodontais, adolescentes, teste de BANA, ELISA.
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Correspondence: Walter A. Bretz, University of Pittsburgh, School of Dental Medicine, Division of Restorative Dental Sciences, 3501 Terrace Street, Pittsburgh, PA 15261, USA. Tel: +1-41-648-8087, FAX: +1-412-353-7796. E-mail: firstname.lastname@example.org
Accepted March 13, 2000
Eletronic publication July, 2000