CLINICAL FEATURES, AGE AND SEX DISTRIBUTIONS, RISK FACTORS AND THE TYPE OF BACTERIA ISOLATED IN PERIODONTITIS PATIENTS IN SANA'A, YEMEN

Background: Periodontitis is an inflammation caused by plaque in the surrounding dental structures. It is a major factor in adult tooth loss. There is lack of information on associated clinical features, risk factors and microbial etiology of periodontitis in Sana’a, Yemen. 
Aim: The study focused on associated clinical features, risk factors and the separation and classification of bacteria in periodontitis and associated risk factors amongst patients attending dental clinics is Sana’a city.  
Methods: First, 296 patients were admitted to the dental clinic at the Republican University Hospital and private dental clinics in Sana'a during a period of nearly one year, which began in December 2019 AD and ended in November 2020 AD, when they were diagnosed with dental diseases, then 49 of them were selected who are suffering from periodontitis, of whom 22 are males and 27 are females. Sterile paper points were used for the sample collection. Standard culture and biochemical techniques were used for the isolation and identification. Structured questionnaires were used to record clinical features, demographic variables and other risk factors of periodontitis. 
Results: A total of 130 microorganisms were isolated from 49 patients with periodontitis. Male patients accounted for 14.4% and females 18.9% of the all 296 patients who attended our clinics.  There was no important association between sex and periodontitis occurrence while there was significant association the younger age groups (45.4% in <26 years of age). The most common signs and symptoms were swollen or puffy gums (91.9%), bleed easily gums (96%), halitosis (96%), painful chewing (87.8%), pus between teeth and gums (71.4%), loose teeth or loss of teeth (44.9%), gingival recession (83.7%), spitting out blood when brushing or flossing teeth (79.6%), and tender gums (93.9%). 
Conclusion: This study is new in Sana'a city. The clinical features of preiodentitis in Yemen and the risk factors are similar to those reported in the literature elsewhere, but the isolated bacteria differ in frequency from those reported elsewhere, as some upper respiratory tract pathogens such as Streptococcus pyogenes are commonly isolated in this study. Knowledge of the clinical features, bacterial causes of gum disease, and risk factors is the key to successful periodontal therapy. 
                    
Peer Review History: 
Received 8 January 2021; Revised 5 February; Accepted 25 February, Available online 15 March 2021 
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Received file:                Reviewer's Comments: 
Average Peer review marks at initial stage: 6.0/10 
Average Peer review marks at publication stage: 7.5/10 
Reviewer(s) detail: 
Dr. Tamer Elhabibi, 
Suez Canal University, Egypt, tamer_hassan@pharm.suez.edu.eg 
Dr. Rawaa Souhil Al-Kayali, 
Aleppo University, Syria, rawah67@hotmail.com 
Rola Jadallah,  
Arab American University, Palestine, rola@aauj.edu 
Prof. Dr. Gorkem Dulger, 
 Duzce University, Turkey, gorkemdulger@yandex.com 
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INTRODUCTION
There is a significant spread of periodontal disease worldwide, and identify the etiology is the solution to controlling it. Periodontal disease is characterized by a chronic bacterial infection with persistent inflammation, breakdown of connective tissue and destruction of the alveolar bone 1 . Generally periodontal disease is classified into periodontitis and gingivitis. Gingivitis is inflammation of the gums initiated by the proliferation of dental plaque and is reversible. Gingivitis is possibly common in children up to 5 years old 2 . It is the result of inappropriate oral hygiene practices 3 . Periodontitis is a chronic inflammatory disease that begins with a buildup of dental biofilm plaque and continues through a disorganized immune response and is commonly started by gingivitis leading to irreversible demolition of the supportive tissues adjoining the tooth, together with the alveolar bone 1,2,4 . Periodontal disease is a multi-microbial, multifactorial disease, with numerous host factors involved in influential an individual's susceptibility to disease 5 . Several reports have been description that the onset and sequence of the disease is not only similar to the existence of pathogenic bacterial strains in the gums but also due to the absence or minimum levels of beneficial equivalents commensals in the susceptible host 5-7 . A limited number of periodontal pathogens have been reported in the complex biofilms to initiate periodontal disease. Data clarified that some bacterial strains in the gingival environment can cause gingivitis and bone destruction. These bacterial strains are known as periodontal pathogens 8, 9 . It is known that these periodontal disease pathogens possess, when present in even very small quantities, the capability to damage the gingival structure 5 . It is known that most periodontal pathogens are anaerobes, but biofilm can also accommodate facultative aerobes, capnophiles, and microaerophiles whose number depends on the environment in the developing biofilms and gingival pocket 5 . It became known that of 800-1000 species that colonized the oral cavity, 50 species have been identified with strong links to periodontal disease 10 . These complexes were classified Socratic and Hafidian into 5 complexes, which are the yellow or early colonized complex, the green or secondary colonized complex, then the orange, purple and red complexes. The red compound is the secondary colony that is the major pathogens related with bleeding upon investigation 11 . There is strong bacterial progression in oral cavity infection that may be predisposed by age, diet, or site of infection 12 . Nevertheless, in addition to periodontal disease pathogens, genetic and environmental factors predispose to disease progression. The risk of gum disease is determined by several factors including any health condition that leads to bacterial defense mechanisms defect such as diabetes, human immunodeficiency virus (HIV) and neutropenia. Tobacco smoking, Obesity, poor diet, and a inactive lifestyle are associated with an increased risk of periodontitis 13 . In the early stages, periodontitis has few symptoms, and the disease has progressed significantly in many individuals before they seek treatment. Symptoms may include redness or bleeding of the gums while brushing the teeth, the use of dental floss or gnawing on solid food, frequent swelling of the gums, gingival recession gingival recession, halitosis, deep pockets between the teeth and the gums, loose teeth, and drifting of incisors 1,3,14 . Periodontitis is the leading cause of tooth loss in adults universally and these people are at risk of edentulism, multiple tooth loss and masticatory impairment as a result of which a negative impact on nutrition, quality of life and self-esteem and thus a significant societal imposition -the economic impact and the cost of health care 3,14,15 . Most of the information about the causes of periodontitis emerged from studies conducted in Europe and the United States of America, and some third world countries. Although there are some studies on oral and dental problems in Yemen 16-25 , no study has been conducted in Yemen on periodontitis 26 . So this study focused on associated clinical features, risk factors and the isolation and identification of bacteria in periodontitis and associated risk factors among patients attending some dental clinics is Sana'a city.

Patients
This study included 49 patients suffering from periodontitis, who were admitted to the dental clinic at the Republican University Hospital and private dental clinics (Al-Mortadda dental clinics, Al-Abany dental clinics and Al-Kahara dental clinics) in Sana'a, during a period of about one year, which started in December 2019 and ended in November 2020, of whom 22 were males and 27 were females. Data collection and microbial processing A questionnaire was filled out for each patient with the patient's personal, clinical data and risk factors. This included age, gender, occupation and relevant clinical information regarding bacterial oral infections. Also risk factors of contracting periodontitis. Cultures were obtained from the collected pocket by probes in order to isolate the various bacterial causative agents. First, the supragingival plaque was removed (without disturbing the subgingival plaque) and a bacterial sample was collected from the deepest periodontal pockets with a sterile probe. The samples were then placed in a vial containing 2 ml of liquid thioglycolate enriched medium, sealed immediately and transported to the laboratory within 30 minutes. Bacteriological procedures were performed within one hour of sample collection. For germ cultures, the following media and conditions were used: Tryptic Soy Agar (TSA) with blood (5%) and MacConkey agar plates -incubated at 35°C under 5% CO 2 and examined at 24 and 48 hours; Brucella agar enriched with Vitamin K1 and CDC + amikacin blood agar -incubated at 35°C anaerobically in a Gaspak jar (Oxoid Ltd). Cultures were examined for the presence of bacteria at 48 and 96 h. Plates showing bacterial growth were retained until final processing and organism identification by classical standard techniques including culture colonies morphology, microscopy staining methods, and biochemical tests 13, 27 .

Data analysis
Clinical, personal, and risk factors data as well as sample culture results entered into the questionnaire were analyzed by Epi Info, Version 6. All subjects with pockets less than 2 mm were considered to have periodentitis. To correlate the clinical features and potential risk factors for periodentitis, the data were examined in the form of case-control studies. For people with periodentitis, people with other dental diseases have been matched. Differences in categorical variables were assessed using Fisher's exact tests as appropriate. Ninety-five percent confidence intervals for odds ratios were calculated according to the Cornfield limits and 95% confidence intervals were calculated for simple ratios by an exact binomial method. The significance of the difference in the ratio and the odds ratio was analyzed, and a chi-square (χ2) greater than 3.84 and a probability value (p) less than 0.05 were considered statistically significant.

Ethical approval
Ethical approval was obtained from the Medical Research and Ethics Committee of the Faculty of Medicine and Health Sciences at Sana'a University. All data, including patient identification were kept confidential.

RESULTS
Male patients accounted for 14.4% of the total periodontitis patients, and the female percentage was 18.9% from the total dental patients attending to the clinics. Table 1 show the age and gender distribution of patients with periodontitis. There was no significant association between gender and periodontitis occurrence. When age was considered a dependent factor for periodontitis, the rate of periodontitis was highest in the younger age groups (45.4% in <26 years of age), while the rate in> 45 years was 13.3%. Table 2 shows the importance periodontitis signs and symptoms among dental patients in Sana'a, Yemen. When compared periodontitis with other oral diseases, swollen or puffy gums occurred in 91.9% of periodontitis patients and occurred more than 30 Table 3 shows the risk factors associated with periodontitis among dental patients in Sana'a, Yemen.

DISCUSSION
Periodontal disease is an increasing health problem in Yemen. Currently, no work has been done to determine the clinical features, etiological and risk factors for periodontitis in Sana'a City, but little works have been done, they dealt with the spread of oral and dental diseases; and some dental and oral disorders [16][17][18][19][20][21][22][23][24][25] . In the current study, there was no significant association between sex and the incidence of periodontitis, and many other researchers appear to favor the female preference 28, 29 . Also, current work contrasts with that of Ababneh and others, who reported a predisposition to males but current result is similar to that reported by Susin and Albander, which reported an equal distribution 30,31 .  Table 2 such as swollen or puffy gums, bleed easily gums, halitosis, painful chewing, pus between teeth and gums, loose teeth or loss of teeth, gingival recession, spitting out blood when brushing or flossing teeth, and tender gums occur more frequently in periodontitis patients than in patients with other oral diseases. This finding is similar to that reported in the literature in which previous signs and symptoms appeared in periodontitis more than other dental diseases 33-35 . In the current study, bleeding gums occurred in 96% of periodontitis patients and occurred 67 times more (CI =15-284, p <0.001) than other oral diseases. In a previous study by Maduakor et al., 36 patients with bleeding gums showed an odds increase of 38.41 times the incidence of gingivitis and 2.58 times the incidence of periodontitis compared to patients without bleeding gums. It has also been reported that bleeding gums is one of the early signs of developing gum disease. This confirms the effect of maintaining good oral health and hygiene associated with preventing inflammation 37, 38 . In the current study higher prevalence of periodontitis among subjects with low education (27.3%) with a significant correlation between a low education level and periodontitis, in which subjects with low school education were 2.9 times more likely to have periodontitis than subjects with a higher level of education (OR=2.9 times, CI=1.5-5.4, p<0.001) ( Table  3). A high prevalence of periodontitis has been reported among low-education patients in Nigeria, Jordan and Thailand 30,36, 39 . This confirmed that in the United States of America, people with a low school education were three times more likely to have periodontitis than people with a higher education level 40 and it has been reported in many studies that there is a correlation between gum disease and educational level. In the current study, patients with a previous history of recurrent gingivitis were approximately 3.8 times more likely to have periodontitis than those without a previous history of gingivitis (OR=3.2, CI=1.7-6.1, p <0.001) ( key pathogens or "red complex" bacterium 2,51 . A. actinomycetecomitans in aggressive chronic periodontitis patients 52 . It is reported to be strongly associated with destructive periodontal lesions 5 . It possesses many virulence factors including protease, leukotoxin, endotoxin, collagenase, fibroblast inhibition factor inducing bone resorption 5 . Staphylococcus aureus was isolated from 53.1% of used periodontitis patients, as did several researchers 52,53 . These microbes are known to easily become resistant to antibiotics, and may reach climax with super-infection. The capability to form biofilm has enabled Staphylococcus aureus to survive in this environment also 49, 54 . Streptococcus pyogenes was isolated from 73.5% and Streptococcus mutans in 16.3% of our patients, and this result is similar to that previously reported in which Streptococcus species was detected in large numbers by several researchers 12,52 . Some streptococci are useful to the host as colonization of the pocket in large numbers can delay the periodontal disease process 55 . In current study, Enterobacteriaceae was isolated in 8% of patients with periodontitis (Table 4). Enterobacteriaceae is unusual in patients with periodontitis 56 . Several studies have linked enteric bacilli to periodontal disease 52 . According to Botero and colleagues, their role in periodontitis is not clear but is thought to indicate super-infection 57 . They are thought to be opportunists that thrive after periodontal treatment. The drug of choice for the treatment of periodontal disease includes tetracycline, doxycycline, amoxicillin, and metronidazole. The gut bacteria illustrate resistance to these drugs and may consequently persist after taking them. Further studies are needed to explain its presence in the plaque biofilm and explain its role in periodontal infection 57 . Candida albicans was isolated in current study in 8% of patients with periodontitis, and several researchers including Daniluk et al., have reported that Candida albicans could have a role in the ultrastructure of gingival microbial plaques and in their attachment to periodontal tissue 25,58,59 .

CONCLUSION
This study is new in Sana'a city. The clinical features of periodontitis in Yemen and the risk factors are similar to those reported in the literature elsewhere, but the isolated bacteria differ in frequency from those reported elsewhere, as some upper respiratory tract pathogens such as Streptococcus pyogens are commonly isolated in this study. Knowledge of the clinical features, bacterial causes of gum disease, and risk factors is the key to successful periodontal therapy.

AUTHOR'S CONTRIBUTION
All authors participated in overseeing clinical and laboratory work, data analysis, and manuscript writing and review.