INTRODUCTION

Periodontitis before the age of 20 is rare. Susin et al. [1] reported that aggressive forms of periodontitis occurred in less than 0.5% of the world population in a review mainly including adolescents, with significant differences between populations.

Studies on the prevalence of advanced periodontitis in juvenile populations worldwide report a quite low prevalence among Caucasians, higher in Hispanic and Asian populations, and most prevalent in African populations [1-7]. A recent review on the global prevalence of periodontitis, which involved studies only in young individuals, estimated a pooled prevalence of 4.2% in Africa compared to 0.1% in Europe [8]. Studies from Africa have mainly been performed in populations geographically located in West Africa (e.g., Ghana, Morocco), while little is known about populations from other parts of Africa.

Periodontitis in children and adolescents has long been associated with A. actinomycetemcomitans, a Gram-negative facultative short rod [9]. This bacterial species was suggested as a putative periodontal pathogen due to its unique ability to produce a leukotoxin [10]. Later, a highly toxic genotype (the JP2 clone) was recognized, which was comparatively prevalent in juvenile periodontitis cases of West African populations [6, 11, 13], whereas extremely rare in Caucasians. The current knowledge about the distribution of this clone in other African populations is very limited; only one case study from Ethiopia [14] was identified in our literature search. Two cases were also detected among immigrants from North-African immigrants in Germany [15]. Since the referred studies on microbiology have focused mainly on A. actinomycetemcomitans, knowledge of other periodontally associated bacteria among African populations is limited [12, 16, 17].

Somali immigrants have in the last decades become a significant immigrant group in Swedish society and according to the Central Bureau of Statistics in Sweden, 70,000 inhabitants were born in Somalia (https://www.scb.se/en/). In a study on oral conditions and background factors in Somali immigrants newly arrived in Sweden, Svensson et al. [18] reported a prevalence of periodontitis of 12% in Somali adolescents aged 11–17 years in Kronoberg county. If this high prevalence in Somali individuals is confirmed, this strongly indicates a need for the dental caregiver to prepare to meet the needs of the changing population as well as emphasizing the need for research to better understand the nature of periodontitis development in young African immigrants.

We therefore decided to carry out a clinical and microbiological study in Somali immigrants and compare them with Swedish subjects of non-Somali heritage of the same age in order to evaluate the prevalence of periodontitis as well as the prevalence and number of A. actinomycetemcomitans (and the JP2 clone in particular) and some other periodontitis-associated bacterial species.

MATERIAL AND METHODS

This study was approved by the Ethical Review Board in Gothenburg, Sweden (Dnr 551-16 and 1064-18). Two groups of participants, 10–17-year-old children and adolescents, on a recall program at the clinic of Sylte, Trollhättan, Public Dental Health Services, Region Västra Götaland, were invited to participate in the study. The two groups consisted of 100 subjects of Somali heritage (Somali group) and 100 Swedish subjects of non-Somali heritage (non-Somali group). Informed consent was signed by the parents of the participants. All information concerning the study was presented in Somali and/or Swedish. The recruitment of the participants for the Somali group was made by author NA, native born Somali dentist, educated in Sweden, who phoned the families of the patients with Somali names from the clinic's patient list and confirmed their ethnicity. Similarly, parents of subjects with non-Somali names were contacted by phone and invited to the study by author M.Al. and M.Ay. Totally, 83 Somali and 96 non-Somali subjects agreed to participate and were clinically and microbiologically examined.

Clinical and radiographical examination

The clinical examination was performed in conjunction with a regularly scheduled check-up at the clinic Sylte in Trollhättan. Presence or absence of plaque and calculus was recorded dichotomously at all fully erupted incisors and first molars at four sites/tooth by visual inspection and probing along the gingival margin. Bleeding on probing was also recorded using a graded periodontal probe for incisors and first molars as well as for teeth exhibiting radiographic signs of bone loss. Data from the periodontal examination, age, gender, and information on descent (Somali/non-Somali heritage) were recorded for each patient.

Two to four digitalized intraoral bitewing radiographs were obtained in each patient, one or two on each side of the molar region depending on the eruption of the second molar, using a Focus X-ray unit (Instrumentarium Dental PaloDex). A conventional technique using a Twix fin (Directa) was used to position the film when the bitewing was exposed with 5–7 degrees of angulation. All sites of all teeth depicted in the bitewing radiograph, with an identifiable cemento-enamel junction (CEJ) and alveolar bone crest (BC) were assessed, using the caliper provided in the Romexis (Planmeca) viewer software. The vertical distance between CEJ and BC was measured in all molar and premolar teeth depicted in the radiographs. A consensus discussion was made for 10 cases between each of the authors N.A., M.Al., and M.Ay. with author S.T.-M., who is a specialist in periodontology. Teeth with at least one site reaching more than 3 mm were recorded as having bone loss. None of the participants exhibited indication for radiographic examination of the incisors at the clinical examination, that is, there were no clinical signs of pathology such as deep periodontal pockets.

A patient was diagnosed as having periodontitis when exhibiting at least one site with clear radiographic signs of bone loss (CEJ-BC > 3 mm) and gingival inflammation as indicated by bleeding on probing (BoP) [19]. Due to sometimes large amounts of plaque and calculus and profuse bleeding tendency, probing pocket depth was not taken into consideration.

Microbiological analysis Sampling

Pooled samples were taken from the mesial subgingival site of four teeth (16, 11, 31, and 36) with a curette corresponding to 1 μl (1 μl inoculation loop; Sarstedt) and was transferred to transport medium VMGAIII (Viability Medium; anaerobically prepared and sterilized according to Dahlén et al. [20]). All samples were transported to the Laboratory of Oral Microbiology, Institute of Odontology, University of Gothenburg for microbiological analysis and were processed within 48 h.

Microbiological analysis by culture

The transport medium (VMGAIII) samples were thoroughly mixed and 100 μL was diluted in a tenfold dilution series. A volume of 100 μL of the concentrated sample from each dilution was spread evenly onto one Brucella (Becton Dickinson) blood agar plate supplemented with 1.5% hemolyzed human erythrocytes, 5 × 105 mg/L menadione, and incubated anaerobically in jars for calculations of the total number of viable counts (TVC), fusobacteria and black-pigmented anaerobic rods, Campylobacter species (corroding), and Capnocytophaga (gliders) species. The Brucella agar plates were cultured anaerobically for 7 days at 36°C. The total number of the specific bacteria was counted. Fusobacterial species were identified as grayish nacreous colonies and filamentous Gram-negative rods on Gram staining. Black-pigmented colonies with Gram-negative rods were identified based on their fluorescence in UV-light either as Porphyromonas gingivalis (non-fluorescent) or Prevotella intermedia/Prevotella nigrescens (brick red fluorescence) [21]. Gram-negative rods with a colony morphology with a spread and corroding appearance and showing motility were classified as Campylobacter spp., while those with a gliding appearance were classified as Capnocytophaga species. The detection level with culture was > 1000 CFU/mL at best.

Furthermore, one TSBV agar plate (trypticase-soy agar with bacitracin 75 mg/L and vancomycin 5 mg/L) was inoculated according to Slots [22] for selective culture of Aggregatibacter actinomycetemcomitans. The plates were incubated for 5 days at 37℃ and 10% CO2. The plates were inspected for colonies with typical A. actinomycetemcomitans morphology showing star-shaped or small smooth, adherent, and catalase-positive colonies, and with a micro-morphology being Gram-negative coccobacilli. The detection level of A. actinomycetemcomitans on TSBV was 10 CFU/mL transport medium.

Microbial quantification with qPCR

The samples in VMGAIII were also analyzed with real-time polymerase chain reaction (qPCR) using 16S specific primers according to Kuboniwa et al. [23] against Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, and A. actinomycetemcomitans. Briefly, the samples were washed with tris-buffer, vortexed and centrifuged 3 times, and then boiled for 10 min. Bacterial genomic DNA was extracted from 200 μL of each sample using the QIAmp DNA Mini-Kit (Qiagen). The PCR assay was performed in 10 μl of LightCycler 480 Probes Master (Roche Diagnostics), forward and reverse primers at 0.25 μM each, 0.25 μM Taqman probe, and 5 μl of bacterial DNA sample. The cycling protocol includes an enzyme activation step at 94°C for 10 min, followed by 40 cycles of amplification, 95°C for 15 s, and 60°C for 1 min.

The presence of the JP2 clone of A. actinomycetemcomitans was identified separately from VMGAIII-extracted DNA using specific primers of the leukotoxin gene (ltxA) using the method of Claesson et al. [24], and positive reaction in qPCR was confirmed by electrophoresis. Detection level was calculated to be 40 counts per 5 μL DNA sample.

Statistics

The proportions of teeth exhibiting BoP/plaque/calculus among all examined teeth (incisors and first molars) were calculated for each patient. Next, mean values and standard deviations were calculated for each group of patients.

The prevalence of bone loss was calculated and the 95% confidence interval (CI) for this estimate was calculated using an online calculator (https://sample-size.net/confidence-interval-proportion/)

Differences between Somali and non-Somali participants in the frequency of bacterial species were statistically evaluated using the chi-square test, while differences in the number of bacterial species was evaluated using Mann-Whitney U-test. Differences with a probability of 5% (P < 0.05) were considered statistically significant.

RESULTS

Ten Somali subjects were diagnosed with periodontitis,that is, they exhibited the radiographic finding of a CEJ-BC distance > 3 mm and BoP. This corresponded to a prevalence of 12% (95% CI: 5.9, 21.0%) among the Somali, while periodontitis was not found in any of the non-Somali participants (Table 1). Bone loss was found in one to three nonadjacent teeth in the ten subjects in the premolar and molar region. Molar teeth were the most frequently affected teeth. The mean age of the Somali participants with periodontitis was higher (15.7 years) than the mean of the Somali group (13.8 years) and the total mean age of all subjects (13.5 years). The Somali participants exhibited more bleeding, plaque, and calculus than the non-Somali. Bleeding was a frequent finding in both groups and the mean percentage of examined teeth with BoP was 100% in the Somali and 75.2% in the non-Somali participants. Plaque and calculus were abundant in the Somali group (on average 100% and 78.3% of teeth, respectively), while they were lower in the non-Somali group (46.7% and 37.5%, respectively).

TABLE 1. Clinical and radiographic findings of Somali and non-Somali children and adolescents Variable Somali Non-Somali Total number of subjects 83 96 Gender, number of female subjects 41 (49.4%) 51 (53.1%) Mean age (range) yr 13.8 (10–17) 13.3 (10–17) BoP*, mean (± SD) 100% 75.2±26.3% Plaque*, mean (± SD) 100% 46.7±32.1% Number of subjects (%) with Calculus 65 (78.3%) 36 (37.5%) Number of subjects (%) with a periodontitis diagnosis† 10 (12.0%) 0 (0%) *Proportion of teeth exhibiting BoP/Plaque out of all examined teeth (incisors and first molars) for each patient, mean values and standard deviations. †Periodontal diagnosis based on radiographic finding of CEJ-BC > 3 mm and BoP.

Table 2 shows that 17 (21.8%) Somali subjects had A. actinomycetemcomitans, while this bacterial species was detected in only one of the non-Somali subjects (P < 0.001) by culture. P. intermedia/P. nigrescens showed a higher frequency (P < 0.001) in Somali subjects (51.4% vs. 28.1%), while 48 (57.8%) of the Somali subjects had detectable Fusobacterium spp. with culture compared to 81 (84.4%) of the non-Somali (P < 0.001). The presence and number of Capnocytophaga was at the same level in both groups (Table 2). Corroding bacterial colonies could not be identified due to the numerous colonies with a gliding motility. Campylobacter species were therefore not recorded from culture.

TABLE 2. Number of positive samples and frequency (%) of 5 bacterial species detected by culture in subgingival samples from 83 Somali and 96 non-Somali study participants Bacteria Somali N = 83 (%) Non-Somali N = 96 (%) P-value A. actinomycetemcomitans 17 (21.8)* 1 (1.04) <0.001 P. gingivalis 1 (1.2) 0 (0) 0.281 P. intermedia/P. nigrescens 51 (69.9)† 27 (28.1) <0.001 Fusobacterium spp. 48 (57.8) 81 (84.4) <0.001 Capnocytophaga spp. 39 (47.0) 52 (54.2) 0.338 Chi-square test was used for statistical calculations (P-value) of bacterial frequency. *n = 78, due to overgrowth of Gram-negative enterics on TSBV agar. †n = 73. Missing data on 10 Somali participants.

With qPCR, the Somali participants demonstrated a strongly statistically significant (P < 0.001) higher frequency and numbers of A. actinomycetemcomitans, P. gingivalis, and T. denticola (Table 3 and Figure 1 A, B, D). The frequency of P. intermedia, T. forsythia, and F. nucleatum was similarly high (86.5-100%) in both groups of participants (Table 3), while the numbers (counts) in positive samples were significantly higher (P < 0.001) for P. intermedia (Figure 1E) and F. nucleatum (Figure 1F) in the Somali group, and significantly (P < 0.001) higher for T. forsythia (Figure 1C) in the non-Somali group.

TABLE 3. Number of positive subjects and frequency (%) of six bacterial species and the JP2 clone of A. actinomycetemcomitans identified using real-time polymerase chain reaction in subgingival samples from 83 Somali and 96 non-Somali study participants Bacteria Somali N = 83 (%) Non-Somali N = 96 (%) P-value A. actinomycetemcomitans 41 (49.4) 0 (0) <0.001 P. gingivalis 80 (96.4) 2 (2.1) <0.001 T. forsythia 82 (98.8) 87 (90.6) 0.018 T. denticola 65 (78.3) 8 (8.3) <0.001 P. intermedia 83 (100) 83 (86.5) <0.001 F. nucleatum 80 (100)* 88 (91.7) 0.008 JP2 clone 5 (6.0) 0 (0) 0.015 Chi-square test was used for statistical calculations (P-value) of bacterial frequency. *n = 80. Missing data on three Somali participants.

Bacterial counts (counts per 2 μL DNA sample) for each individual analyzed by specific qPCR in positive samples (detection level 40 counts per sample) from Somali and non-Somali children, with differences in the number of species evaluated using the Mann-Whitney U-test. (A) A. actinomycetemcomitans (P = 0.288). (B) P. gingivalis (P = 0.340). (C) P. intermedia (P < 0.001). (D) T. forsythia (P < 0.001). (E) T. denticola (P < 0.167). (F) F. nucleatum (P < 0.001).

The JP2 clone was detected in five (6.0%) of the samples from Somali subjects, two from cases with periodontitis, and three from cases with non-detectable periodontitis. The JP2 clone was not detected in any of the samples from the non-Somali subjects.

DISCUSSION

The present study compared 83 Somali immigrant children and adolescents with 96 non-Somali Swedish peers of similar age (10–17 years) clinically, radiographically, and microbiologically for the prevalence of periodontitis and the presence and numbers of periodontitis marker bacteria.

Despite the young age, 12% of the Somali participants were diagnosed with periodontitis while none of the non-Somali exhibited bone-loss to the extent of >3 mm on radiographs. This figure corresponds to the findings of Svensson et al. [18] examining Somali children living in Kronoberg county, Sweden, using the same radiographic parameters and ages. Considering that the study population consists solely of children and adolescents, participants who presented only a single site with bone loss and BoP were also viewed as having periodontitis in the present study. Five of the ten participants with bone loss exhibited two or more sites at nonadjacent teeth, fulfilling the definition of a periodontitis case according to Tonetti et al. [19].

There is considerable variation in case definitions and few studies are using both clinical and radiographical measurements to ascertain periodontitis. When including both clinical and radiographic signs of the disease, the prevalence of periodontitis has been reported to be between 0.1% and 0.8% [16],[25]. A prevalence of periodontitis was found in 2%–4.5% of Scandinavian 7 to 14-year-old children using a distance of >2 mm between CEJ and alveolar BC as a sign of disease, based on radiographs alone [26],[27]. It has been suggested that a distance between CEJ-BC within 0–2 mm represents healthy status [28],[29]. The radiographic threshold of >3 mm was chosen in the present study to discriminate periodontitis from gingivitis in combination with the clinical finding of BoP, because the intention was to identify clearly diseased cases. In a clinical study on 12–21-years-olds in Chile [30] it was found that 70% had at least one site with CEJ-bottom of pocket ≥ 1 mm, while a distance ≥2 mm was found in 16% and a distance ≥3 mm in 4.5%. This demonstrates a substantial variation in prevalence dependent on the threshold used. Sjödin and Matsson [29] reported a mean value of CEJ-BC on 7–9 year-olds and the normal range of 0.0–2.0 mm and considered CEJ-BC of ≥2 mm to be pathological, supported by Sardana et al. [28] reporting that distances of 2.5 mm or more should be considered disease.

Gingival inflammation represented by BoP was a common finding in both groups. A striking difference between the groups found during examination was the poor oral hygiene among most of the Somali participants with plaque and calculus to an extent and abundance not seen in any of the non-Somali subjects. The children were recruited in a random fashion and should therefore represent the real conditions of the two ethnic groups. The Somali children were natively born in Somalia and had immigrated to Sweden within the last 10 years, many of them within the last year. The abundance of plaque and inflammation was indeed a risk factor in the Somali group but may not solely explain the finding of periodontitis at such a young age. It rather indicates a cultural neglect of oral hygiene routines in the Somali population and emphasize the urgence of treatment and immediate need for improved oral hygiene and health.

Although gingival inflammation and plaque is a prerequisite for the development of periodontitis, the relationship is not linear. In an early study in a community with poor oral hygiene, represented by general high levels of plaque, calculus, and bleeding, a proportion of the population was more affected by periodontitis, introducing the concept of individual susceptibility [31]. A wide range of host-related risk factors such as heredity, diabetes, psychological factors, and smoking have been identified but do not fully explain the expression of disease [32].

While children and adolescents often exhibit plaque and gingivitis [33], onset of periodontitis is an uncommon finding before the age of 20 years [9]. Using radiographic and clinical findings, Saxén et al. reported a prevalence of 0.1% in a study of 8069 16-year-olds in Finland [34]. In a study on 8666 children in Sweden 7-, 8-, and 9-year-olds were examined by radiographic examination alone. Bone loss > 2 mm was detected at one or more sites with a prevalence of 2.0%, 3.1%, and 4.5%, respectively, for the 7-, 8-, and 9-year-olds [5]. In this present study the Somali children and adolescents exhibited a high prevalence of periodontitis. Similar results (prevalence of 12%) were seen in the study on Somali immigrated youngsters (11–17-years-old) in Kronoberg county [18], confirming the comparatively higher susceptibility for periodontitis in the Somali population.

The microbiological analysis was made with culture and with qPCR. Culture was performed in order to isolate and purify bacterial strains for further use, knowing the lower specificity and sensitivity for most bacterial species using culture. Using selective media (TSBV-agar), we found a significantly higher prevalence of A. actinomycetemcomitans among the Somali children, while the prevalence and number of P. intermedia/P. nigrescens, Fusobacterium spp., and Capnocytophaga spp. using non-selective media was equally high in both subject groups. It should be noted that both methods, culture on selective media and qPCR, should be considered sensitive, with qPCR being even more sensitive than culture. However, they do not measure the same thing—culture measures viable and growing cells as colony forming units (CFU), while qPCR detects species-specific DNA including even dead cells and free DNA.

Using qPCR, the significantly higher prevalence of A. actinomyctemcomitans in Somali children was confirmed. In five of the children, it was also possible to identify the JP2 clone. This pattern thus resembles the picture of West-African populations in the literature [6, 13, 35], and indicates that the JP2 clone may be spread more widely over the African continent. The strong association between A. actinomycetemcomitans (and the JP2 clone in particular) with periodontal disease was confirmed in this Somali population, however longitudinal follow-up may disclose whether this bacterial species and the JP2 clone prospectively constitute risk markers for further progression in these children and have a pathological impact on periodontitis as was proposed for Ghanian children by Höglund Åberg et al. [35].

The frequency of P. gingivalis and T. denticola was significantly higher in the Somali subjects than the non-Somali among whom these two bacterial species was detected in only a few cases. Whilst these two bacteria have been associated with periodontitis in adults [36] in many populations worldwide [37, 38], they are less studied in children or adolescents. Although P. gingivalis can be detected in children, even at ages as low as 4 years, it is suggested to be consistently present and established after puberty [39, 40]. The low frequency of P. gingivalis and T. denticola in the non-Somali children may indicate population/ethnic differences also for other microorganisms and subtypes thereof than A. actinomycetemcomitans and the JP2 clone. It seems more likely, however, that due to insufficient tooth cleaning procedures and accumulation of calculus, the Somali children and adolescents develop an undisturbed subgingival microbiota, which resembles a longstanding and matured microbial ecology seen in adults.

T. forsythia was found in a high frequency in both subject groups. However, the number was surprisingly and significantly higher in the non-Somali participants despite less plaque and gingivitis and no detectable periodontal breakdown. This may reflect a true difference in the development of a periodontal microbiota between the populations. The microbial ecology and composition may not meet the necessary requirements for growth of bacterial species such as T. forsythia. Colonization of T. forsythia has been associated with the presence of teeth rather than periodontitis [40] and a prevalence of 25% was detected among English adolescents without periodontitis [41]. Therefore, T. forsythia does not serve well as marker bacteria for periodontitis in adolescents (juveniles).

Other periodontitis-associated bacteria, such as P. intermedia, F. nucleatum and Capnocytophaga spp. all belonging to the orange complex according to Socransky et al. [36], were found in high frequency and numbers both with culture and with qPCR in the two groups. These bacterial species are consistently found in all populations worldwide. They are established early in life and although they are considered late colonizers of the dental plaque [42], they are frequently found even in the mucosa and saliva of individuals demonstrating ‘healthy gingiva’. These bacteria belong to the resident human oral microbiota and are well adapted to the subgingival environment in which they increase their growth and numbers as a result of increasing inflammation.

Children and adolescents of Somali heritage clearly showed a more extensive disease pattern of gingivitis and periodontitis compared to similarly aged children and adolescents of non-Somali heritage. Although an ethnically related increased susceptibility to periodontitis in young Somali individuals cannot be excluded, the difference in disease pattern was overshadowed by the poor or absent oral hygiene routines resulting in abundant plaque and calculus deposits among the Somali and distinct differences in the specified subgingival microbiota between the two groups. Due to this, targeted efforts are recommended in the form of increased vigilance to detect early signs of disease during regular examinations and intensified motivation and instruction in oral hygiene measures when required. When the demographic panorama changes due to a more globalized world, dental care providers need to adapt their diagnostic attention and provide structures for care and prevention to match the needs in our changing local society.

In conclusion, the Somali participants showed a more extensive periodontal disease pattern than the non-Somali control group and 12% had radiographically visible bone loss (>3 mm). The presence of A. actinomycetemcomitans was almost exclusively associated with the Somali participants. Further, the JP2 clone was found in five Somali participants (two were periodontitis cases) confirming the association of this clone to African populations. The Somali group showed significantly higher frequency and number of Porphyromonas gingivalis and Treponema denticola, implying a more mature and adult type of subgingival microbiota than that of the non-Somali.

ACKNOWLEDGEMENTS

The authors want to thank the Eva Magnusson and all personnel at the Public Dental health Clinic at Sylte, Trollhättan, Sweden for letting us perform this study. We specially want to thank also dentist Suzanne Baromand, for excellent assistance in practical matters at the clinic. We are grateful also to Susanne Blomqvist and Anna-Karin Östberg for technical assistance in the laboratory.

The study was supported by the Public Dental Health Service (Folktandvården), VGregion, Sweden. The study was further supported by a student grant from the Institute of Odontology and the laboratory costs were covered by the Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Sweden.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

AUTHOR CONTRIBUTIONS

Conceptualization: Sara Thorbert-Mros, Anna Trullenque-Eriksson, Gunnar Dahlen. Investigation: Sara Thorbert-Mros, Nawal Ali, Meis Ali, Mert Ayas, Gunnar Dahlén. Formal analysis: Sara Thorbert-Mros, Anna Trullenque-Eriksson, Gunnar Dahlén. Writing—original draft: Nawal Ali, Meis Ali, Mert Ayas. Writing—review and editing: Sara Thorbert-Mros, Anna Trullenque-Eriksson, Gunnar Dahlén.