Natural Bioactive Compounds in the Management of Periodontal Diseases: A Comprehensive Review.

Periodontal diseases, chronic inflammatory conditions affecting oral health, are primarily driven by microbial plaque biofilm and the body's inflammatory response, leading to tissue damage and potential tooth loss. These diseases have significant physical, psychological, social, and economic impacts, necessitating effective management strategies that include early diagnosis, comprehensive treatment, and innovative therapeutic approaches. Recent advancements in biomanufacturing have facilitated the development of natural bioactive compounds, such as polyphenols, terpenoids, alkaloids, saponins, and peptides, which exhibit antimicrobial, anti-inflammatory, and tissue regenerative properties. This review explores the biomanufacturing processes-microbial fermentation, plant cell cultures, and enzymatic synthesis-and their roles in producing these bioactive compounds for managing periodontal diseases. The integration of these natural compounds into periodontal therapy offers promising alternatives to traditional treatments, potentially overcoming issues like antibiotic resistance and the disruption of the natural microbiota, thereby improving patient outcomes.

Effectiveness of Diode (810 nm) Laser in Periodontal Parameters and Reduction of Subgingival Bacterial Load in Periodontitis Patients.

AIM: This split-mouth randomized trial (RCT) aimed to assess the effect of diode laser on the clinical parameters in patients with periodontitis, compare the results with scaling and root planing (SRP) alone, and assess the implications of diode laser (DL) on plaque bacteria. MATERIALS AND METHODS: Seventeen periodontitis patients were randomly assigned into two equal groups based on the therapy delivered. Group I (control site) received just SRP at baseline, while group II (test site) received both SRP and DL irradiation. For both groups, the clinical periodontal parameters probing pocket depth (PPD), and clinical attachment level (CAL) were measured at baseline, 30 days, and 90 days. Microbiological amount was also measured at baseline, 30, and 90 days after periodontal treatment. The amounts of Aggregatibacter actinomycetemcomitans (A.a), Prevotella intermedia (Pr. intermedia), and Porphyromonas gingivalis (P. gingivalis) were determined using real-time PCR probing with specific bacterial primers. RESULTS: In both groups, PPD and CAL showed statistically significant reductions at different time intervals (p < 0.05). No significant difference were observed in CAL values after 1 and 3 months in both test and control groups (p > 0.05). The mean values of the concentration of A.a, Pr. intermedia and P. gingivalis were lower in the case group as compared to the control group and the difference was statistically significant after 1 month (*p = 0.001). CLINICAL SIGNIFICANCE: According to this study, non-invasive laser treatment has the potential to improve clinical outcomes by lowering the quantity of A.a, Pr. intermedia and P. gingivalis. CONCLUSION: In both groups, a considerable decrease in the periodontal pathogens A.a, Pr. intermedia and P. gingivalis were discovered; however, the intergroup comparison was insignificant in relation to PD and CAL. The adjunctive treatment with diode laser showed better efficacy in ensuring a better periodontal treatment than SRP alone. How to cite this article: Abdullah LA, Hashim N, Rehman MM, et al. Effectiveness of Diode (810 nm) Laser in Periodontal Parameters and Reduction of Subgingival Bacterial Load in Periodontitis Patients. J Contemp Dent Pract 2023;24(12):1008-1015.

Soluble Receptor Activator of Nuclear Factor Ligand and Osteoprotegerin Levels in Gingival Crevicular Fluid among Cigarette Smokers and Non-smokers with and without Periodontitis.

AIM: This study aimed to measure and compare the levels of soluble receptor activator of nuclear factor ligand (RANKL) and osteoprotegerin (OPG) in the gingival crevicular fluid (GCF), as well as their ratio, in smokers and nonsmokers with periodontitis. MATERIALS AND METHODS: Gingival crevicular fluid samples were collected using PerioPaper strips, from 150 individuals, who were categorized into three groups: current smokers with periodontitis stage III grades C and B (n = 50), nonsmokers with periodontitis stages I and II grade A (n = 50), and control healthy individuals (n = 50). The concentrations (pg/mL) of sRANKL and OPG in the GCF were measured by enzyme-linked immunesorbent assays (ELISA). RESULT: The smokers' group exhibited the highest sRANKL (pg/mL) concentration as a subsequent lead to a higher sRANKL/OPG ratio. The healthy control group exhibited higher OPG and lower sRANKL concentration, subsequently, the sRANKL/OPG ratio was reduced compared with the other study groups. However, there was no statistical significance of sRANKL and its relative ratio between periodontitis stage III grades C and B, periodontitis stages I and II grade A, and healthy control individuals. There was a statistically significant positive moderate correlation between smoking duration (years) and the sRANKL (pg/mL) concentration and a statistically significant negative moderate correlation between OPG (pg/mL) concentration and cigarettes smoked per day. CONCLUSION: As a result, compared to the other research groups, smokers with periodontitis stage III grades C and B had greater GCF concentrations of sRANKL, lower OPG, and a higher sRANKL/OPG ratio. The difference in OPG (pg/mL) level was statistically significant. However, there was no statistically significant difference in sRANKL (pg/mL) or its relative ratio, sRANKL/OPG, across the groups. CLINICAL SIGNIFICANCE: A characteristic that sets periodontitis apart is alveolar bone loss. Resorption is induced by RANKL and inhibited by OPG, resulting in a relative ratio. In light of this, the levels of RANKL and OPG may be helpful indicators for monitoring the activity of periodontal disease in both smokers and nonsmokers with and without periodontitis.

Replication of the association of GLT6D1 with aggressive periodontitis in a Sudanese population.

BACKGROUND: Susceptibility to aggressive periodontitis (AgP) is influenced by genetic as well as environmental factors. Studies linking gene variants to AgP have been mainly centred in developed countries with limited data from Africa. AIM: To investigate whether previously reported candidate gene associations with AgP could be replicated in a population from Sudan. METHODS: The investigation was a case-control design. Cases with AgP (n = 132) and controls (n = 136) were identified from patients attending the Periodontal Department in Khartoum Dental Hospital. Genotyping was performed using the Sequenom MassARRAY iPLEX platform. Analysis focused on gene variants with a minor allele frequency (MAF) > 25% in the Sudanese subjects that had previously been reported to be associated with AgP. RESULTS: One candidate gene rs1537415 (GLT6D1) was significantly associated with AgP, OR = 1.50 (95% CI 1.04-2.17), p = 0.0295 (increasing to p = 0.09 after correction for multiple testing). The association strengthened to OR = 1.56 (95% CI 1.15-2.16), p = 0.0042 when the controls were supplemented with data from the Hap map for the Yoruba in Ibadan (n = 147) and remained significant (p = 0.013) after correction for multiple testing. CONCLUSION: The study independently replicated the finding that rs1537415, a variant in glycosyl transferase gene GLT6D1, is associated with AgP and provided the first report of genetic associations with AgP in a Sudanese population.

Highlighting the Effect of Pro-inflammatory Mediators in the Pathogenesis of Periodontal Diseases and Alzheimer's Disease.

Alzheimer's disease (AD) is a neurological condition that is much more common as people get older. It may start out early or late. Increased levels of pro-inflammatory cytokines and microglial activation, both of which contribute to the central nervous system's inflammatory state, are characteristics of AD. As opposed to this, periodontitis is a widespread oral infection brought on by Gram-negative anaerobic bacteria. By releasing pro-inflammatory cytokines into the systemic circulation, periodontitis can be classified as a "low-grade systemic disease." Periodontitis and AD are linked by inflammation, which is recognized to play a crucial part in both the disease processes. The current review sought to highlight the effects of pro-inflammatory cytokines, which are released during periodontal and Alzheimer's diseases in the pathophysiology of both conditions. It also addresses the puzzling relationship between AD and periodontitis, highlighting the etiology and potential ramifications.

NLRP3 Inflammasome in Autoinflammatory Diseases and Periodontitis Advance in the Management.

Inflammatory chemicals are released by the immune system in response to any perceived danger, including irritants and pathogenic organisms. The caspase activation and the response of inflammation are governed by inflammasomes, which are sensors and transmitters of the innate immune system. They have always been linked to swelling and pain. Research has mainly concentrated on the NOD-like protein transmitter 3 (NLRP3) inflammasome. Interleukin (IL)-1 and IL-18 are pro-inflammatory cytokines that are activated by the NOD-like antibody protein receptor 3 (NLRP3), which controls innate immune responses. The NLRP3 inflammasome has been associated with gum disease and other autoimmune inflammatory diseases in several studies. Scientists' discovery of IL-1's central role in the pathophysiology of numerous autoimmune disorders has increased public awareness of these conditions. The first disease to be connected with aberrant inflammasome activation was the autoinflammatory cryopyrin-associated periodic syndrome (CAPS). Targeted therapeutics against IL-1 have been delayed in development because their underlying reasons are poorly understood. The NLRP3 inflammasome has recently been related to higher production and activation in periodontitis. Multiple periodontal cell types are controlled by the NLRP3 inflammasome. To promote osteoclast genesis, the NLRP3 inflammasome either increases receptor-activator of nuclear factor kappa beta ligand (RANKL) synthesis or decreases osteoclast-promoting gene (OPG) levels. By boosting cytokines that promote inflammation in the periodontal ligament fibroblasts and triggering apoptosis in osteoblasts, the NLRP3 inflammasome regulates immune cell activity. These findings support further investigation into the NLRP3 inflammasome as a therapeutic target for the medical treatment of periodontitis. This article provides a short overview of the NLRP3 inflammatory proteins and discusses their role in the onset of autoinflammatory disorders (AIDs) and periodontitis.

Gum Arabic as a potential candidate in quorum quenching and treatment of periodontal diseases.

Periodontal diseases are chronic inflammatory conditions influenced by bacterial biofilm formation and host immune responses, affecting millions worldwide. Traditional treatments like mechanical debridement and systemic antibiotics often face limitations, including biofilm resilience and antibiotic resistance. Gum Arabic (GA), a natural exudate from Acacia trees, presents a promising alternative with its anti-biofilm and anti-inflammatory properties. This review highlights the role of GA in periodontal therapy, particularly its ability to interfere with quorum sensing (QS) pathways, specifically the AI-2 signaling system used by key periodontal pathogens such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. By disrupting QS, GA inhibits biofilm formation, reduces bacterial virulence, and promotes a balanced oral microbiome. GA's prebiotic properties also encourage the growth of beneficial bacteria, enhancing the host's immune response while preserving the systemic microbiome. Clinical studies demonstrate GA's effectiveness as an adjunct in periodontal therapy, with significant reductions in plaque accumulation, gingival inflammation, and bleeding. This highlights GA's potential as a natural therapeutic agent, offering an effective, antibiotic-sparing option in managing periodontal disease. However, further research is warranted to fully establish GA's role in comprehensive periodontal care and its long-term benefits.

Knowledge and Preparedness of Dentists in Response to Bioterrorism.

Introduction: In the current climate of heightened terrorist activity, the deliberate release or threat of biological agents like viruses, bacteria, fungi, or their toxins to induce sickness or death among civilians has become a genuine possibility. The most crucial action during a bioterrorist strike is early warning. The possibility of such an occurrence in dental setting is underestimated. Methods and Materials: Seventy five graduate students participated in the research. In a survey on zoonotic diseases, participants were asked whether they were familiar with the transmission from person to person, if the diseases caused significant morbidity and mortality, whether they were simple to produce and disseminate, and whether they posed a high risk of infection. Results: Graph Prism was used to examine the data. A single proportion test was used to determine the "Aware Not Aware" group. The first 10 questions assessed dentists objective knowledge of bioterrorism. The importance of the last six questions was determined by their analysis of dentists' preparedness and desire to respond to a bioterrorism associated occurrence via perceived knowledge. Conclusion: Given the real danger they may face, dentists needed to be vigilant regarding the prevention, detection, and treatment of zoonotic illnesses transmitted in their offices or as a vector while being commended for their courage in the face of adversity.

A New Scoring System of Oral Lichen Planus.

Lichen planus (LP) is a chronic, immune-mediated mucocutaneous disorder increasingly becoming common in the general population with female predominance. Clinically, there are different forms of lichen planus with the presence of the main characteristic feature of Wickham striae. Literature, to date, is abundant with various scoring systems of oral lichen planus, and among them, the most commonly followed scoring system was the one proposed by the Thongprasom system because of its simplicity and ease of application. Aim: The aim of the present study is to critically review all the disease scoring systems on oral lichen planus (OLP) that have been reported in the literature during the past decades. A systematic literature search was performed using PUBMED, MEDLINE, EMBASE, and COCHRANE Library with language restriction to English. The search was carried out incorporating the published literature from 1980 to 2020 using the MeSH (medical subject heading) terms. A literature search was done using keywords: Staging, Grading, Oral lichen planus, Diagnostic, and Therapeutic. Out of 25 publications, related to search strategy, 22 full articles, which were related to the disease scoring system for oral lichen planus, were acquired for further inspection. Out of the 22 articles, 15 articles met the inclusion criteria. The data was collected and a brief summary of the studies regarding the different disease scoring systems for oral lichen planus was explained. Taking into consideration, the parameters were not included in the previous disease scoring system. A new proposal encompassing a scoring system for oral lichen planus considering the missing parameters along with an amalgamation of histopathological criteria of dysplasia is presented. It also proposes to grade and stage the lesions and recommend appropriate therapy for each of such lesions.

Therapeutic Switching of Rafoxanide: a New Approach To Fighting Drug-Resistant Bacteria and Fungi.

Control and management of life-threatening bacterial and fungal infections are a global health challenge. Despite advances in antimicrobial therapies, treatment failures for resistant bacterial and fungal infections continue to increase. We aimed to repurpose the anthelmintic drug rafoxanide for use with existing therapeutic drugs to increase the possibility of better managing infection and decrease treatment failures. For this purpose, we evaluated the antibacterial and antifungal potential of rafoxanide. Notably, 70% (70/100) of bacterial isolates showed multidrug resistance (MDR) patterns, with higher prevalence among human isolates (73.5% [50/68]) than animal ones (62.5% [20/32]). Moreover, 22 fungal isolates (88%) were MDR and were more prevalent among animal (88.9%) than human (87.5%) sources. We observed alarming MDR patterns among bacterial isolates, i.e., Klebsiella pneumoniae (75% [30/40; 8 animal and 22 human]) and Escherichia coli (66% [40/60; 12 animal and 28 human]), and fungal isolates, i.e., Candida albicans (86.7% [13/15; 4 animal and 9 human]) and Aspergillus fumigatus (90% [9/10; 4 animal and 5 human]), that were resistant to at least one agent in three or more different antimicrobial classes. Rafoxanide had antibacterial and antifungal activities, with minimal inhibitory concentration (MICs) ranging from 2 to 128 µg/mL. Rafoxanide at sub-MICs downregulated the mRNA expression of resistance genes, including E. coli and K. pneumoniae blaCTX-M-1, blaTEM-1, blaSHV, MOX, and DHA, C. albicans ERG11, and A. fumigatus cyp51A. We noted the improvement in the activity of ß-lactam and antifungal drugs upon combination with rafoxanide. This was apparent in the reduction in the MICs of cefotaxime and fluconazole when these drugs were combined with sub-MIC levels of rafoxanide. There was obvious synergism between rafoxanide and cefotaxime against all E. coli and K. pneumoniae isolates (fractional inhibitory concentration index [FICI] values ≤ 0.5). Accordingly, there was a shift in the patterns of resistance of 16.7% of E. coli and 22.5% of K. pneumoniae isolates to cefotaxime and those of 63.2% of C. albicans and A. fumigatus isolates to fluconazole when the isolates were treated with sub-MICs of rafoxanide. These results were confirmed by in silico and mouse protection assays. Based on the in silico study, one possible explanation for how rafoxanide reduced bacterial resistance is through its inhibitory effects on bacterial and fungal histidine kinase enzymes. In short, rafoxanide exhibited promising results in overcoming bacterial and fungal drug resistance. IMPORTANCE The drug repurposing strategy is an alternative approach to reducing drug development timelines with low cost, especially during outbreaks of disease caused by drug-resistant pathogens. Rafoxanide can disrupt the abilities of bacterial and fungal cells to adapt to stress conditions. The coadministration of antibiotics with rafoxanide can prevent the failure of treatment of both resistant bacteria and fungi, as the resistant pathogens could be made sensitive upon treatment with rafoxanide. From our findings, we anticipate that pharmaceutical companies will be able to utilize new combinations against resistant pathogens.

Rhamnolipid-Coated Iron Oxide Nanoparticles as a Novel Multitarget Candidate against Major Foodborne E. coli Serotypes and Methicillin-Resistant S. aureus.

Surface-growing antibiotic-resistant pathogenic bacteria such as Escherichia coli and Staphylococcus aureus are emerging as a global health challenge due to dilemmas in clinical treatment. Furthermore, their pathogenesis, including increasingly serious antimicrobial resistance and biofilm formation, makes them challenging to treat by conventional therapy. Therefore, the development of novel antivirulence strategies will undoubtedly provide a path forward in combatting these resistant bacterial infections. In this regard, we developed novel biosurfactant-coated nanoparticles to combine the antiadhesive/antibiofilm properties of rhamnolipid (RHL)-coated Fe3O4 nanoparticles (NPs) with each of the p-coumaric acid (p-CoA) and gallic acid (GA) antimicrobial drugs by using the most available polymer common coatings (PVA) to expand the range of effective antibacterial drugs, as well as a mechanism for their synergistic effect via a simple method of preparation. Mechanistically, the average size of bare Fe3O4 NPs was ~15 nm, while RHL-coated Fe3O4@PVA@p-CoA/GA was about ~254 nm, with a drop in zeta potential from -18.7 mV to -34.3 mV, which helped increase stability. Our data show that RHL-Fe3O4@PVA@p-CoA/GA biosurfactant NPs can remarkably interfere with bacterial growth and significantly inhibited biofilm formation to more than 50% via downregulating IcaABCD and CsgBAC operons, which are responsible for slime layer formation and curli fimbriae production in S. aureus and E. coli, respectively. The novelty regarding the activity of RHL-Fe3O4@PVA@p-CoA/GA biosurfactant NPs reveals their potential effect as an alternative multitarget antivirulence candidate to minimize infection severity by inhibiting biofilm development. Therefore, they could be used in antibacterial coatings and wound dressings in the future. IMPORTANCE Antimicrobial resistance poses a great threat and challenge to humanity. Therefore, the search for alternative ways to target and eliminate microbes from plant, animal, and marine microorganisms is one of the world's concerns today. Furthermore, the extraordinary capacity of S. aureus and E. coli to resist standard antibacterial drugs is the dilemma of all currently used remedies. Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) have become widespread, leading to no remedies being able to treat these threatening pathogens. The most widely recognized serotypes that cause severe foodborne illness are E. coli O157:H7, O26:H11, and O78:H10, and they display increasing antimicrobial resistance rates. Therefore, there is an urgent need for an effective therapy that has dual action to inhibit biofilm formation and decrease bacterial growth. In this study, the synthesized RHL-Fe3O4@PVA@p-CoA/GA biosurfactant NPs have interesting properties, making them excellent candidates for targeted drug delivery by inhibiting bacterial growth and downregulating biofilm-associated IcaABCD and CsgBAC gene loci.

In Situ Biosynthesis of Reduced Alpha Hematite (α-Fe2O3) Nanoparticles by Stevia Rebaudiana L. Leaf Extract: Insights into Antioxidant, Antimicrobial, and Anticancer Properties.

In the present study, we utilized Stevia rebaudiana L. (SRLe) extract to in situ biosynthesize nanoscale alpha hematite (α-Fe2O3) nanoparticles (NPs) with potent antioxidant, antimicrobial, and anticancer properties. SRLe-α-Fe2O3 was characterized using physiochemical analyses, including UV/Vis, FTIR, XRD, DLS, EDX, SEM, and TEM studies. Among tested solvents, CHCl3/MeOH (2:1 v/v) SRL extract (least polar solvent) contained the highest EY, TPC, and antioxidant capacity of ~3.5%, ~75 mg GAE/g extract, and IC50 = 9.87 ± 0.7 mg/mL, respectively. FTIR confirmed the engagement of coating operation to the colloidal α-Fe2O3 NPs. TEM, SEM, and DLS revealed that SRLe-α-Fe2O3 has a spherical shape, uniform size distribution with aggregation for an average size of ~18.34 nm, and ζ = -19.4 mV, forming a repulsive barrier that helped to improve stability. The synthesized nanoparticles displayed considerable antibacterial activity against E. coli and S. aureus bacterial growth, and exhibited superior activity against the A549 lung cancer cell lines. These findings indicate that the increased availability of bioactive substances with antioxidant properties of SRLe makes it a potentially interesting material for the preparation of biologically active compounds and green synthesis of nanoparticles.

Salivary TNFα levels in groups of subjects with rheumatoid arthritis and chronic periodontitis.

BACKGROUND: Rheumatoid arthritis (RA) and chronic periodontitis are the most common chronic inflammatory diseases with significant pathological and clinical similarities. Numerous studies have indicated a relationship between rheumatoid arthritis and periodontal disease. The aim of this study was to compare the TNF-α levels in saliva among patients with Rheumatoid arthritis (RA) and chronic periodontitis as well as healthy subjects. METHODS: One hundred and seventy-one patients were enrolled in this cross-sectional study. Fifty-seven patients diagnosed of RA, 57 patients with chronic periodontitis and 57 healthy subjects. These patients have been examined with regard to TNF-α level from salivary samples. Their teeth were examined with regard to Plaque Index , Gingival Index, probing depth and clinical attachment level.All patients were non-smokers. RESULTS: The results revealed a significant difference in all periodontal parameters among the three groups. The chronic periodontitis group showed a significantly higher value in all clinical periodontal parameters in comparison to both the RA and healthy groups. No significant difference was found between salivary TNF-α level among the three study groups. CONCLUSIONS: Patients with chronic periodontitis had the highest periodontal indices. However there was no significant difference regarding the level of salivary TNF-α. Hence, suppression of proinflammatory cytokines might prove beneficial in suppressing periodontal diseases among RA patients.

Effect of the clinical application of the diode laser (810 nm) in the treatment of dentine hypersensitivity.

BACKGROUND: Dentine hypersensitivity is a common clinical finding with a wide variation in prevalence values. The aim of this study was to evaluate the use of diode laser (810 nm) in the treatment of cervical dentine hypersensitivity. METHODS: Five patients, with at least two sensitive teeth were selected. A total of 14 teeth were included in this trial. By using Visual Analogous Scale the pain of dentine hyper sensitivity was detected and the pre- treatment readings were recorded.The Diode laser (810 nm), was irradiated on (non contact) mode at the cervical region.The samples were divided into two groups according to exposure duration: For Group 1 exposure duration was 30 seconds and for group 2 exposure duration was one minute.The efficiency of the treatment was assessed at two examination period :15 minutes after first application and 7 days after first application, the degree of sensitivity was determined by using Visual Analogous Scale. RESULTS: The results show significant reduction of pain after 15 minutes of laser application in the group with 30 seconds exposure duration (P = .001), and the pain completely fade away after one week in the same group, while in the group with 1 minute exposure duration the pain completely disappeared (visual analogous scale = (0)) after 15 minutes and one week of laser application (P = 0.001). CONCLUSION: The study concluded that application of diode laser (810 nm) was effective for the reduction of dentine hypersensitivity.

Periodontal status of rheumatoid arthritis patients in khartoum state.

BACKGROUND: Few studies have investigated the periodontal condition among Rheumatoid arthritis in Sudan. The present study described the periodontal condition among Sudanese patients suffering from rheumatoid arthritis and to compare them with those of non-rheumatic subjects. METHODS: A group of eighty rheumatoid arthritis patients was selected from Patient's Rheumatoid Clinics in Khartoum State during the period of January to May 2010. A control group of eighty patients with the same age and gender was selected for the study. Both Rheumatoid arthritis patients and the control group were examined for their plaque index, gingival index, and clinical attachment loss. RESULTS: The results revealed that there were no significant differences in plaque and gingival index among study and control groups, with mean plaque index of (1.25 ± 0.4) for patients and (1.17 ± 0.28) for the control group (p-value is 0.3597). The mean gingival index was (1.2 ± 0.24) for the patients and (1.2 ± 0.33) for the control (p = is 0.3049). The results showed statistically significant differences in clinical attachment loss between study and control groups, with mean clinical attachment loss of (1.03 ± 0.95) for the study group and (0.56 ± 0.63) for the control group (p = 0.0002). The study revealed that no association exists between the type of drug used to treat rheumatoid arthritis (NSAIDs & DMARDs) and the periodontal parameters (plaque index, gingival index, and clinical attachment loss). CONCLUSION: A significant relationship between periodontal disease and Rheumatoid Arthritis does exist, but no difference between plaque and gingival index has been detected among study and control groups.