Supra- and Subgingival Microbiome in Gingivitis and Impact of Biofilm Control: A Comprehensive Review.

This comprehensive review aimed (1) to characterize the sub- and supragingival microbiome in patients with biofilm-induced gingivitis (including experimental gingivitis), (2) to assess its stability and evolution over time, and (3) to assess the impact of biofilm control measures on this stability. An electronic search of the MEDLINE®/PubMed® database until December 2023 was conducted. NCBI Taxonomy, eHOMD 16S rRNA Reference Sequence, and Tree Version 15.23 databases were used to standardize taxonomic nomenclature. Out of 89 papers initially retrieved, 14 studies were finally included: 11 using experimental gingivitis as a model and three randomized clinical trials evaluating the impact of biofilm control measures. Among them, five characterized the subgingival microbiome, nine the supragingival microbiome, and one both the sub- and supragingival microbiome. In addition, five studies evaluated the effect of toothpaste, and four studies evaluated the effect of mouth rinses. The diversity and structure of the microbiome differed significantly between patients with periodontal health and those with biofilm-induced gingivitis (including experimental gingivitis). Those differences were not reversed through conventional oral hygiene measures. Specific antiseptic agents, especially if delivered as mouth rinses, may have an impact on the supra- and subgingival microbiome in gingivitis.

Domains, competences and learning outcomes for undergraduate education in periodontology.

AIM: This review is intended to adapt the current conceptual framework in dental education based on four domains to propose a set of competences, learning outcomes and methods of teaching, learning and assessment for undergraduate education in periodontology. REVIEW: Based on the current framework of competences and learning outcomes recommended by the Association for Dental Education in Europe (ADEE), undergraduate education in periodontology has been updated using the classification and clinical practice guidelines for the diagnosis and treatment of periodontal and peri-implant diseases. CONCLUSIONS: Specific learning outcomes have been proposed within each competence area, that is in Domain I (n = 10), Domain II (n = 13), Domain III (n = 33) and Domain IV (n = 12). Teaching methods and learning activities based on the different dimensions of the cognitive process have been proposed. Additionally, 10 key learning outcomes have been proposed as exit outcomes, which implies their accomplishment within the final assessment of any graduating student.

Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis.

OBJECTIVE: This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. METHODS: The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established. RESULTS: obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R2: 0.997-0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%-72.73; kappa 0.37-0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2-100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1). CONCLUSIONS: The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test.

Differential analysis of culturable and unculturable subgingival target microorganisms according to the stages of periodontitis.

OBJECTIVES: Culturable and unculturable microorganisms have been associated with periodontitis. Their differential proportions and composition have not been evaluated by their severity and complexity defined by stages in the 2018 AAP-EEP classification. METHODS: One hundred eighty subgingival biofilm samples were collected in Spain and Colombia from subjects categorized as health/gingivitis: periodontitis stages I/II periodontitis stages III/IV. Target culturable microorganisms (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Eubacterium nodatum) and target unculturable microorganisms (Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis) were evaluated by quantitative PCR analysis. In addition, their differences and association with periodontal status were analyzed by ANCOVA and logistic regression models once adjusted to age, current smoking, and country. RESULTS: P. gingivalis was significantly associated with periodontitis stages I/II, OR 2.44 (CI 95% 1.08-5.47) and stages III/V, OR 6.43 (CI 95% 2.43-16.9). T forsythia, OR 7.53 (CI 95% 2.07-27.4); D. oralis, OR 5.99 (CI 95% 2.71-13.23); F. alocis, OR 10.9 (CI 95% 4.56-23.2); E. brachy, 3.57 (CI 95% 1.40-9.11); and E. saphenum, 4.85 (CI 95% 1.99-11.7) were significantly associated only with stages III/IV periodontitis. P. gingivalis evidenced significant differences with the increase in the severity of the periodontal lesion: 2.97 colony forming unit (CFU)/µL (CI 95% 2.32-3.54) health/gingivitis, and 4.66 CFU/µL (CI 95% 4.03-5.30) and 5.90 CFU/µL (CI 95% 5.20-6.48) in stages I/II and III/IV respectively (p < 0.0001). Unculturable microorganisms only evidenced differences in concentration in stages III/IV compared with health-gingivitis (p ≤ 0.001). CONCLUSION: Culturable and unculturable are strongly associated with stages III/IV periodontitis. Classic culturable microorganisms are more sensitive to differentiate between stages of periodontitis in the quantitative analysis. CLINICAL RELEVANCE: Future interventional studies of periodontal disease should include Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, and Desulfobulbus oralis as possible markers of therapy response and as indicators of progressive disease.

Subgingival microbiome in periodontal health, gingivitis and different stages of periodontitis.

AIM: To characterize the subgingival microbiome in subjects with different periodontal health statuses. MATERIALS AND METHODS: In this cross-sectional observational study, subgingival samples were harvested from Spanish subjects with different periodontal health statuses, based on the 2018 Classification of Periodontal and Peri-Implant Diseases and Conditions. Samples were processed using high-throughput sequencing technologies (Illumina MiSeq). Taxa differentially abundant were identified using Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC). α- and ß-diversity metrics were calculated using q2-diversity in QIIME2. The analyses were adjusted for age, gender and smoking status. RESULTS: The identified subgingival microbiome showed statistically significant differences among subjects, categorized into periodontal health, gingivitis and stages I-II and III-IV periodontitis (p < .05). In patients with severe (stages III-IV) periodontitis, the genera Filifactor and Fretibacterium were detected 24 times more frequently than in periodontally healthy subjects. Similarly, the genera Porphyromonas, Prevotella and Tannerella were detected four times more frequently (p < .05). The genera Granulicatella, Streptococcus, Paracoccus, Pseudomonas, Haemophilus, Actinobacteria, Bergeyella and Capnocytophaga were significantly associated with healthier periodontal status (p < .05). CONCLUSIONS: Significant differences were detected in the subgingival microbiome among periodontal health, gingivitis and stages I-II or III-IV periodontitis, suggesting overlapping, yet distinguishable microbial profiles.

Dental Biofilm Removal and Bacterial Contamination of a New Doubled-Side Thermoplastic Polyurethane-Based Toothbrush: A Crossover Study in Healthy Volunteers.

Multiple toothbrush designs have been developed to enhance dental biofilm removal and decrease bacterial contamination and retention over time. Therefore, the aim of this clinical study was to compare the efficacy of a prototype of a new double-sided thermoplastic polyurethane-based toothbrush with that of a conventional nylon-bristle toothbrush. A crossover study was conducted in systemically healthy volunteers (n = 24) for two one-week periods plus one washout week. As outcome variables, plaque and gingival indices, total bacterial contamination of the toothbrushes by quantitative polymerase chain reaction (qPCR), and patient-reported outcomes were measured. Clinical and microbiological variables were analysed using a general linear model and Friedman and Wilcoxon signed-rank tests. No statistically significant differences between toothbrushes were detected neither for full-mouth PlI (p > 0.05) nor for GI (p > 0.05). Similarly, no statistically significant differences were detected for bacterial contamination after 40 seconds or 1 week of use, with results expressed either in CFU/mL or in CFU/mm2 (p > 0.05). In conclusion, the tested prototype toothbrush was as effective and safe as the control toothbrush, and the participating subjects did not experience any adverse effects from its use and rated its efficiency and effectiveness in cleaning their teeth as satisfactory.

Differences in the subgingival microbiome according to stage of periodontitis: A comparison of two geographic regions.

No microbiological criteria were included in the 2018 EFP-AAP classification of periodontal diseases that could be used to differentiate between stages and grades. Furthermore, differences in the subgingival microbiome depending on stage and grade have not been established. Sixty subgingival biofilm samples were collected in Spain (n = 30) and Colombia (n = 30) from three distinct patient categories: those with periodontal health/gingivitis (n = 20), those with stage I-II periodontitis (n = 20), and those with stage III-IV periodontitis (n = 20). Patients were evaluated by 16S rRNA gene amplification sequencing. Amplicon sequence variants were used to assign taxonomic categories compared to the Human Oral Microbiome Database (threshold ≥97% identity). Alpha diversity was established by Shannon and Simpson indices, and principal coordinate analysis, ANOSIM, and PERMANOVA of the UNIFRAC distances were performed using QIIME2. Although differences in the alpha diversity were observed between samples according to country, Filifactor alocis, Peptostreptococcaceae [XI][G-4] bacterium HMT 369, Fretibacterium fastidiosum, Lachnospiraceae [G-8] bacterium HMT 500, Peptostreptococcaceae [XI][G-5] [Eubacterium] saphenum, Peptostreptococcus stomatis, and Tannerella forsythia were associated with periodontitis sites in all stages. However, only F. alocis, Peptostreptococcaceae [XI][G-4] bacterium HMT 369, Peptostreptococcaceae [XI][G-9] [Eubacterium] brachy, Peptostreptococcaceae [XI][G-5] [Eubacterium] saphenum, and Desulfobulbus sp. HMT 041 were consistent in stage III-IV periodontitis in both countries. Porphyromonas gingivalis and Tannerella forsythia were differentially expressed in severe lesions in the countries studied. Although some non-cultivable microorganisms showed differential patterns between the different stages of periodontitis, they were not the same in the two countries evaluated. Further studies using larger samples with advanced next-generation techniques for high-throughput sequencing of phyla and non-cultivable bacteria within the subgingival microbiome could provide more insight into the differences between stages of periodontitis.

The subgingival cultivable bacteria of Albanian subjects with different periodontal status compared to a similar population of Spanish subjects: a case control study.

BACKGROUND: The objective was to qualitatively and quantitatively describe the subgingival cultivable bacteria in Albanian subjects and to compare it with a similar Spanish population. MATERIALS AND METHODS: Consecutive patients, diagnosed as periodontitis in stages I-II or III-IV, and as periodontally healthy or with gingivitis, were studied clinically and microbiologically by means of microbiological culture, including total anaerobic counts, proportions, and frequency of detection of target species. Outcome variables were analysed by Mann-Whitney, Kruskal-Wallis, ANOVA, ANCOVA and Chi-square tests. RESULTS: In this cross-sectional study, 83 (Albania) and 90 (Spain) subjects were included. No statistically significant differences were observed between test and control populations regarding demographic variables or smoking habit. Significantly higher total anaerobic counts in the Albanian population (p = 0.022) were observed, especially in the periodontal health/gingivitis group (p = 0.001). In the test population, the proportions of the cultivable bacteria of Fusobacterium nucleatum were significantly lower in both the healthy/gingivitis (p = 0.022) and stages I-II periodontitis (p = 0.034) groups. CONCLUSIONS: The subgingival cultivable bacteria in both periodontitis and non-periodontitis subjects from Albania showed significantly higher total anaerobic counts and lower proportions of the cultivable bacteria of F. nucleatum than a similar population of subjects from Spain.

Characterization of the Subgingival Cultivable Microbiota in Patients with Different Stages of Periodontitis in Spain and Colombia. A Cross-Sectional Study.

The objective was to characterize and compare the subgingival microbiota in patients diagnosed according to the World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions 2018. For this cross-sectional study, Spanish and Colombian subjects (characterized as health/gingivitis, periodontitis in stages I-II or stages III-IV) were clinically assessed, and subgingival samples were taken and processed by culture. The comparisons among patients with periodontal status (and between countries) was made using Mann-Whitney, Kruskal-Wallis, ANOVA and chi-square tests. The final sample consisted of 167 subjects. Eikenella corrodens and Parvimonas micra were more frequently detected in health/gingivitis and Porphyromonas gingivalis in periodontitis (p < 0.05). Higher total counts were observed in Colombia (p = 0.036). In Spain, significantly higher levels of P. gingivalis and Campylobacter rectus were observed, and of Tannerella forsythia, P. micra, Prevotella intermedia, Fusobacterium nucleatum, Actinomyces odontolyticus and Capnocytophaga spp. in Colombia (p < 0.001). P. micra was more prevalent in health/gingivitis and stage I-II periodontitis in Colombia, and P. gingivalis in all periodontitis groups in Spain (p < 0.05). As conclusions, significant differences were detected in the microbiota between health/gingivitis and periodontitis, with minor differences between stages of periodontitis. Differences were also relevant between countries, with Colombia showing larger counts and variability of bacterial species.

Análisis de la evolución en el acceso a los medicamentos huérfanos en España / Analysis of the evolution in the access to orphan medicines in Spain

OBJETIVO: Evaluar el acceso al mercado de los medicamentos huérfanos en España que a fecha de 31 de diciembre de 2017 tuvieran vigente su designación, y para aquellos comercializados en España estimar los tiempos entre la asignación de código nacional (CN) por parte de la Agencia Española de Medicamentos y Productos Sanitarios (AEMPS) y la fecha de comercialización efectiva. MÉTODO: La base de datos para identificar los medicamentos huérfanos autorizados por la Agencia Europea de Medicamentos (EMA), a fecha 31 de diciembre de 2017 (n = 142), es su Registro Comunitario publicado por la Comisión Europea. La EMA publica los medicamentos huérfanos que han perdido la designación. Las fechas de asignación de CN provienen de la AEMPS, y las de comercialización, de Bot PLUS. Se llevó a cabo un análisis descriptivo de las variables de estudio. Las variables cuantitativas se describieron utilizando la media y mediana, así como la desviación estándar y su rango. Las variables cualitativas se describieron según frecuencias absolutas y relativas. La comparación de resultados se realizó mediante contrastes paramétricos y no paramétricos en función de la aplicabilidad, con un nivel de significación del 5%. RESULTADOS: Entre 2002 y 2017, la EMA autorizó (con designación vigente a 31 de diciembre de 2017) 100 medicamentos huérfanos. De ellos, 86 tienen CN asignado, y de estos, 54 se han comercializado en España (54% de los medicamentos huérfanos vigentes y 63% de aquellos con CN). Para todos los medicamentos huérfanos con fecha de comercialización (53), el tiempo (mediana) desde la asignación del CN hasta su comercialización en España es de 13,4 meses (desviación estándar: 17,0; mínimo: 2,1; máximo: 91,7). La mediana para los comercializados en 2002-2013 y 2014-2017 es de 12,4 meses y 14,0 meses, respectivamente (p = 0,46). Esta diferencia no es estadísticamente significativa, lo que cabría esperar dado el número limitado de medicamentos huérfanos en nuestra «población». CONCLUSIÓN: Numerosos factores determinan el acceso a los medicamentos huérfanos. La autorización centralizada de comercialización en Europa es un éxito; su acceso es más limitado, dadas las complejidades de evaluación de la evidencia disponible en los procesos de financiación y precio. Es necesario implementar nuevas políticas que reduzcan las desigualdades en el acceso y permitan la sostenibilidad del sistema. Para conseguir estos objetivos, podrían contemplar un proceso acelerado en la decisión de financiación y precio, y el pago por resultados con incertidumbre alta

OBJECTIVE: To assess the access to orphan medicines in Spain, focusing on those with an active "orphan" designation, as of 31st December 2017; and for those orphan medicines in the Spanish market, estimate the time between being assigned a National Code (NC) by the Agencia Española de Medicamentos y Productos Sanitarios (AEMPS) and being approved for launch. METHOD: We used the European Commission's Public Register of orphan medicines to identify the orphan medicines authorised by the European Medicines Agency (EMA), as of 31 December 2017, while we sourced expired orphan indications from the EMA's website. Dates when NCs were assigned were sourced from the AEMPS, and commercialisation dates from Bot PLUS. A descriptive analysis of the study variables was done. The quantitative variables were described using means and medians, as well as standard deviations and ranges. The qualitative variables were described according to absolute and relative frequencies. The comparison of results was performed by parametric and non-parametric contrasts according to the applicability, at a 5% significance level. RESULTS: The EMA has approved 100 orphan medicines (with designation as of 31/12/2017) between 2002-2017. Eighty-six have a NC assigned by the AEMPS. Fifty-four have been launched in Spain (representing 54% of the full sample; 63% with NC). For the 53 orphan drugs with launch date in Spain, the median time between receiving its NC and its launch is 13.4 months (standard deviation: 17.0; minimum: 2.1; maximum: 91,7). The median time is 12.4 months and 14.0 months for those medicines launched in Spain between 2002-2013 and 2014-2017 respectively (p = 0.46). This difference is not statistically significant, which is what could be expected given the low numbers of orphan medicines in the "population". CONCLUSION: Complex factors determine the access to orphan drugs in Europe. The centralised procedure to obtain marketing authorisation at European level is a success. However, access is more limited, given the complexities of the evaluation of the available evidence for pricing and reimbursement decisions. It is therefore necessary to implement new policies that reduce inequalities in access and help achieve sustainable healthcare systems. To achieve this, they will need to offer the possibility of allowing earlier access, and using payment by results when there is high uncertainty

[Analysis of the evolution in the access to orphan medicines in Spain]. / Análisis de la evolución en el acceso a los medicamentos huérfanos en España.

OBJECTIVE: To assess the access to orphan medicines in Spain, focusing on those with an active "orphan" designation, as of 31st December 2017; and for those orphan medicines in the Spanish market, estimate the time between being assigned a National Code (NC) by the Agencia Española de Medicamentos y Productos Sanitarios (AEMPS) and being approved for launch. METHOD: We used the European Commission's Public Register of orphan medicines to identify the orphan medicines authorised by the European Medicines Agency (EMA), as of 31 December 2017, while we sourced expired orphan indications from the EMA's website. Dates when NCs were assigned were sourced from the AEMPS, and commercialisation dates from Bot PLUS. A descriptive analysis of the study variables was done. The quantitative variables were described using means and medians, as well as standard deviations and ranges. The qualitative variables were described according to absolute and relative frequencies. The comparison of results was performed by parametric and non-parametric contrasts according to the applicability, at a 5% significance level. RESULTS: The EMA has approved 100 orphan medicines (with designation as of 31/12/2017) between 2002-2017. Eighty-six have a NC assigned by the AEMPS. Fifty-four have been launched in Spain (representing 54% of the full sample; 63% with NC). For the 53 orphan drugs with launch date in Spain, the median time between receiving its NC and its launch is 13.4 months (standard deviation: 17.0; minimum: 2.1; maximum: 91,7). The median time is 12.4 months and 14.0 months for those medicines launched in Spain between 2002-2013 and 2014-2017 respectively (p = 0.46). This difference is not statistically significant, which is what could be expected given the low numbers of orphan medicines in the "population". CONCLUSION: Complex factors determine the access to orphan drugs in Europe. The centralised procedure to obtain marketing authorisation at European level is a success. However, access is more limited, given the complexities of the evaluation of the available evidence for pricing and reimbursement decisions. It is therefore necessary to implement new policies that reduce inequalities in access and help achieve sustainable healthcare systems. To achieve this, they will need to offer the possibility of allowing earlier access, and using payment by results when there is high uncertainty.

Clinical and microbiological effects of the adjunctive use of probiotics in the treatment of gingivitis: A randomized controlled clinical trial.

AIM: To evaluate the efficacy of a probiotic combination in the treatment of gingivitis and to assess its impact on the subgingival microbiota. MATERIALS AND METHODS: A placebo-controlled clinical trial was conducted in gingivitis subjects during 6 weeks. Test treatment consisted of the administration of two oral tablets per day containing the probiotic strains Lactobacillus plantarum, Lactobacillus brevis and Pediococcus acidilactici; the control group received the same tablets but without live bacteria. The main outcome variable was the changes in gingival index (GI). Subgingival samples were collected and analysed by quantitative polymerase chain reaction (qPCR) for five putative periodontal pathogens. Outcome variables were compared between and within groups, and multiple regression analysis was performed. RESULTS: A total of 59 patients (29 tests, 30 placebos) were included in the analysis. Both treatment groups experienced a statistically significant improvement in mean GI (p < .0001), but no differences between treatment groups were found for any clinical index. A significantly higher reduction in the number of sites with higher GI scores (GI = 3 at baseline) was observed in the test group. In subgingival samples, a significant reduction in T. forsythia was significant only in the test group (p < .008). CONCLUSIONS: The use of probiotic tablets containing L. plantarum, L. brevis and P. acidilactici did not lead to significant changes in mean GI; although a significant reduction occurred in the number of sites with severe inflammation. Furthermore, the adjunctive use of this probiotic promoted a significant microbiological impact.

Antiplaque and antigingivitis toothpastes.

Dentifrices are a general term used to describe preparations that are used together with a toothbrush with the purpose to clean and/or polish the teeth. Active toothpastes were first formulated in the 1950s and included ingredients such as urea, enzymes, ammonium phosphate, sodium lauryl sarcosinate and stannous fluoride. Later, therapeutic agents were included. Today's toothpastes have two objectives: to help the toothbrush in cleaning the tooth surface and to provide a therapeutic effect. The therapeutic effect may have an antiplaque or anti-inflammatory basis when the nature of the agents is antimicrobial. Plaque inhibitory and antiplaque activity of toothpastes used for chemical plaque control is evaluated in distinct consecutive stages, the last being home use randomized clinical trials of at least 6 months' duration. In this chapter, the scientific evidence supporting the use of the most common antiplaque agents, included in toothpaste formulations, is reviewed, with a special emphasis on 6-month clinical trials, and systematic reviews with meta-analyses of the mentioned studies. Among the active agents, the following have been included in toothpastes: enzymes, amine alcohols, herbal or natural products, triclosan, bisbiguanides (chlorhexidine), quaternary ammonium compounds (cetylpyridinium chloride) and different metal salts (zinc salts, stannous fluoride, stannous fluoride with amine fluoride). Dentifrices are the ideal vehicles for any active ingredient used as an oral health preventive measure since they are used in combination with toothbrushing, which is the most frequently employed oral hygiene method. The most important indications of dentifrices with active ingredients are associated with long-term use to prevent bacterial biofilm formation, mostly in gingivitis patients or in patients on supportive periodontal therapy.

Probiotic effects of orally administered Lactobacillus reuteri-containing tablets on the subgingival and salivary microbiota in patients with gingivitis. A randomized clinical trial.

OBJECTIVE: To investigate the effects of an orally administered probiotic on the oral microbiota. METHODS: A placebo-controlled, parallel study was conducted in 40 gingivitis subjects during 8 weeks. Treatment consisted on the administration of a daily tablet, either containing Lactobacillus reuteri or placebo. Unstimulated saliva and subgingival samples were collected and analysed by culture and PCR. Clinical and microbiological outcome variables were compared between and within groups. RESULTS: There were no significant changes between and within the groups in the clinical variables. In saliva, total anaerobic counts after 4 weeks (p = 0.021) and counts of Prevotella intermedia after 8 weeks (p = 0.030), showed reductions in the test group. In subgingival samples, significant reductions in the changes baseline to 4 weeks were observed for P. gingivalis counts (p = 0.008). With PCR, L. reuteri ATCC-PTA-5289 was more frequently detected than L. reuteri DSM-17938. CONCLUSIONS: The effect of L. reuteri administered in tablets resulted in a reduction in the number of selected periodontal pathogens in the subgingival microbiota, without an associated clinical impact.