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

Psoriasis vulgaris: Relationship between oral and periodontal conditions and disease severity

Psoriasis is distributed across the world with a prevalence ranging from 0.5% to 4.6% [1, 2]. This condition has been associated with other diseases, or comorbidities, such as oral cavity involvement, including oral lesions and periodontal disease, thus reaffirming that this is a systemic disease [3, 4]. Psoriasis is a chronic, inflammatory disease which is characterized by the exaggerated proliferation of keratinocytes as a result of immune system activation through T lymphocytes in focal cutaneous areas [5, 6]. In most cases of psoriasis, oral lesions are associated with the presence of geographic and fissured tongue, although data show an increased frequency of associations with generalized pustular psoriasis [7]. It has been estimated that the actual prevalence of fissured tongue and geographic tongue is 6 - 33% and 1-18%, respectively, suggesting that these might represent different expressions of the same disease [7]. However, there is no consensus of the clinical description of what could be considered an oral psoriatic lesion, oral evaluation, or examination, is not a regular procedure in patients with psoriasis [7, 8]. Oral lesions are generally asymptomatic, although there might be tongue swelling and pain when deep fissures are formed [7, 8]. Furthermore, bad tongue hygiene can cause halitosis and tissue swelling due to the accumulation of food residues in fissures, thus causing burning and stinging, especially after contact. However, this association is debatable because it is not known if such oral manifestations are a symptom of disease severity or represent an expression of the disease itself [8]. On the other hand, periodontal disease is marked by an exaggerated response of the immune system to oral microbiota, making it a chronic inflammatory disease which is mediated immunologically, in which immune cells cause inflammation and cellular destruction [9]. Although the two pathologies are similar from the immunological aspect, some characteristics still render them separate diseases and there is no real association known between them [10, 11]. New research has shown that both psoriasis and periodontal disease have both experienced increased prevalence worldwide, and have been associated with other diseases and comorbidities, thus reconfirming that these are, indeed, systemic diseases [12, 13]. There are no existing reports in the published literature which describe the prevalence and frequency of psoriasis and periodontal disease in Colombia. We considered that it was very important for such clinical data to be available because we believe that appropriate examinations should be carried out on all patients with psoriasis, who may have multiple associated comorbidities. We also believe that such examinations should be performed on a regular basis. Furthermore, it is not yet known if the diagnosis of oral psoriasis should be made when lesions in the oral cavity present by themselves, or only at the same time as skin symptoms develop. Consequently, in this study, we aimed to investigate the association between periodontal diagnosis, microbiological components, the presence of IgG against Porphyromonas gingivalis (P. gingivalis), and the clinical manifestations of psoriasis

Hypochlorous Acid as a Potential Postsurgical Antimicrobial Agent in Periodontitis: A Randomized, Controlled, Non-Inferiority Trial

Antecedentes: El ácido hipocloroso (HOCl) es un agente antimicrobiano con alta afinidad por las bacterias Gram negativas del biofilm subgingival. Podría tener un efecto equivalente o no inferior al de la clorhexidina (CHX) para evitar la recolonización de estos microorganismos tras el periodo posquirúrgico. Objetivo: El objetivo es comparar la reducción del índice de placa (IP), el índice gingival (IG), la profundidad de bolsa (PD), la ganancia de nivel de inserción clínica (CAL) y la recolonización bacteriana de microorganismos periodontopáticos en el biofilm subgingival a los 7, 21 y 90 días después del Desbridamiento a Colgajo Abierto (DCA) bajo dos protocolos antimicrobianos: (A) HOCl 0. 05% seguido de HOCl 0,025% y (B) CHX 0,2%/CHX 0,12% utilizados durante 21 días sin higiene bucal regular durante el periodo posquirúrgico. Material y métodos: Se realizó un ensayo controlado aleatorizado de no inferioridad. Treinta y dos pacientes fueron divididos aleatoriamente para recibir cada protocolo antiplaca tras la OFD en pacientes con periodontitis. Se evaluaron los índices clínicos y la recolonización bacteriana mediante qPCR durante 90 días. Los datos se analizaron mediante ANOVA de medidas repetidas, modelos de efectos mixtos ajustados por tratamiento, tiempo y la prueba de Chi-cuadrado/Fisher. También se realizó un análisis de no inferioridad mediante la prueba de hipótesis de no inferioridad de Hodges-Lehmann. Resultados: El HOCl no fue inferior al CHX en la reducción de la IP. Ambos grupos mostraron una reducción comparable de la recolonización de Porphyromonas gingivalis, Tannerella forsythia y Eubacterium nodatum. Sin embargo, el protocolo con HOCl no fue inferior al protocolo con CHX para Treponema denticola y Aggregatibacter actinomicetemcomitans. Conclusiones: El HOCl mejoró la cicatrización periodontal. El HOCl mostró un impacto en la reducción de la recolonización de bacterias periodontopáticas en el postoperatorio. © 2023 por los autores.Background: Hypochlorous acid (HOCl) is an antimicrobial agent with high affinity to Gram-negative bacteria of the subgingival biofilm. It could have an equivalent or no inferiority effect to chlorhexidine (CHX) to avoid recolonization of these microorganisms after the post-surgical period. Objective: The objective is to compare the reduction of plaque index (PI), gingival index (GI), pocket depth (PD), gain of clinical attachment level (CAL), and bacterial recolonization of periodontopathic microorganisms in subgingival biofilm at 7, 21, and 90 days after Open Flap Debridement (OFD) under two antimicrobial protocols: (A) HOCl 0.05% followed by HOCl 0.025% and (B) CHX 0.2%/CHX 0.12% used per 21 days without regular oral hygiene during the post-surgical period. Material and methods: A no-inferiority randomized controlled trial was carried out. Thirty-two patients were randomly divided to receive each antiplaque protocol after OFD in patients with periodontitis. Clinical indexes and bacterial recolonization were assessed using qPCR for up to 90 days. Data were analyzed using repeated measures ANOVA, mixed effects models adjusted for treatment, time, and the Chi-squared/Fisher test. A no-inferiority analysis was also performed using the Hodges–Lehmann hypothesis test for non-inferiority. Results: HOCl was not inferior to CHX in reducing PI. Both groups showed a comparable reduction of recolonization for Porphyromonas gingivalis, Tannerella forsythia, and Eubacterium nodatum. However, the HOCl protocol was non-inferior to the CHX protocol for Treponema denticola and Aggregatibacter actinomicetemcomitans. Conclusions: HOCl improved periodontal healing. HOCl showed an impact in reducing the recolonization of periodontopathic bacteria in the postoperative period. © 2023 by the authors

Niveles elevados de leptina y adipsina se asocian con la actividad clínica en pacientes con artritis reumatoide temprana con sobrepeso e infección periodontal

Las adipocinas están asociadas a la patogénesis de la artritis reumatoide (AR) y son biomarcadores potenciales de la actividad de la enfermedad, la periodontitis y la obesidad. El objetivo era establecer la asociación entre el perfil de adipocinas, la actividad de la enfermedad de la AR, el índice de masa corporal y la infección periodontal. En este estudio se evaluaron 51 pacientes con AR temprana y 51 controles, incluyendo marcadores reumatológicos séricos, niveles de adipocinas, detección de Porphyromonas gingivalis y anticuerpos séricos anti-Porphyromonas gingivalis, y mediciones clínicas y periodontales. Se realizaron análisis estadísticos con SPSS® V26, con un modelo de regresión logística para confirmar las asociaciones. Los resultados muestran que los niveles elevados de leptina eran más frecuentes en pacientes (p = 0,001) que presentaban simultáneamente una mayor frecuencia de Porphyromonas gingivalis (p = 0,004). Los pacientes con presencia concomitante de Porphyromonas gingivalis, alta puntuación de actividad clínica y sobrepeso se correlacionaron con altos niveles de leptina (OR, 7,20; IC 95%, 2,68-19,33; p = 0,0001) y adipsina (OR, 2,69; IC 95%, 1,00-7,28; p = 0,005). La conclusión es que los niveles elevados de leptina y adipsina se asocian a una mayor actividad clínica en pacientes con AR temprana con sobrepeso e infección periodontal, por lo que el sobrepeso y Porphyromonas gingivalis pueden potenciar la actividad de la AR. Esto puede representar un mecanismo patológico entre estas condiciones, donde las adipokinas parecen tener un papel clave.Adipokines are associated with the pathogenesis of rheumatoid arthritis (RA) and are potential biomarkers of disease activity, periodontitis, and obesity. The aim of this was to establish the association between adipokine profile, RA disease activity, body mass index, and periodontal infection. This study evaluated 51 patients with early-RA and 51 controls including serum rheumatological markers, adipokine levels, detection of Porphyromonas gingivalis and serum anti-Porphyromonas gingivalis antibodies, clinical and periodontal measurements. Statistical analyses were run with SPSS® V26, with a logistic regression model to confirm associations. The results show high levels of leptin were more frequent in patients (p = 0.001) who simultaneously showed a higher frequency of Porphyromonas gingivalis (p = 0.004). Patients with concomitant presence of Porphyromonas gingivalis, high clinical activity score, and overweight were correlated with high levels of leptin (OR, 7.20; 95% CI, 2.68–19.33; p = 0.0001) and adipsin (OR, 2.69; 95% CI, 1.00–7.28; p = 0.005). The conclusion is that high levels of leptin and adipsin are associated with greater clinical activity in early-RA patients with overweight and periodontal infection, whereby overweight and Porphyromonas gingivalis may enhance RA activity. This may represent a pathological mechanism between these conditions, where adipokines seem to have a key role

El efecto de interacción de los títulos de anticuerpos anti-RgpA y anti-PPAD: Un indicador para el diagnóstico de la artritis reumatoide

Porphyromonas gingivalis secreta factores de virulencia como Arg-gingipains y peptidil arginina deiminasa (PPAD), que están asociados con la patogénesis de la artritis reumatoide (AR). Sin embargo, no existe información sobre los títulos de anticuerpos frente a estas enzimas bacterianas como indicadores sistémicos o biomarcadores en la AR. En este estudio transversal se evaluó a 255 individuos: 143 con diagnóstico de AR y 112 sin AR. Se utilizaron modelos de regresión logística ajustados por edad, sexo, índice metabólico basal, tabaquismo y gravedad de la periodontitis para evaluar la asociación de la AR con el factor reumatoide (FR), los anticuerpos antiproteínas citrulinadas (ACPA), la velocidad de sedimentación globular, la proteína C reactiva de alta sensibilidad, los anti-RgpA, los anti-PPAD y los anti-RgpA/anti-PPAD doblemente positivos. Se observó que el FR (odds ratio [OR] 10,6; intervalo de confianza [IC] del 95%: 4,4-25), los ACPA (OR 13,7; IC del 95%: 5,1-35) y la doble positividad anti-RgpA/anti-PPAD (OR 6,63; IC del 95%: 1,61-27) se asociaban con el diagnóstico de AR. Los anti-RgpA también se asociaron con la AR (OR 4,09; IC 95%: 1,2-13,9). La combinación de anti-RgpA/anti-PPAD mostró una elevada especificidad del 93,7% y un VPP del 82,5% en la identificación de individuos con AR. Los anticuerpos anti-RgpA se asociaron con el índice inflamatorio periodontal en individuos con AR (p < 0,05). La doble positividad de los anticuerpos anti-RgpA/anti-PPAD mejoró el diagnóstico de AR. Por lo tanto, los anticuerpos RgpA y anti-RgpA/anti-PPAD pueden ser biomarcadores de la AR.Porphyromonas gingivalis secretes virulence factors like Arg-gingipains and peptidyl arginine deiminase (PPAD), that are associated with rheumatoid arthritis (RA) pathogenesis. However, there is no information regarding the antibody titers for these bacterial enzymes as systemic indicators or biomarkers in RA. In this cross-sectional study, 255 individuals were evaluated: 143 were diagnosed with RA, and 112 were without RA. Logistic regression models adjusted for age, sex, basal metabolic index, smoking, and periodontitis severity were used to evaluate the association of RA with rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), erythrocyte sedimentation rate, high sensitivity C-reactive protein, anti-RgpA, anti-PPAD, and double positive anti-RgpA/anti-PPAD. It was found that RF (odds ratio [OR] 10.6; 95% confidence interval [CI] 4.4–25), ACPAs (OR 13.7; 95% CI 5.1–35), and anti-RgpA/anti-PPAD double positivity (OR 6.63; 95% CI 1.61–27) were associated with RA diagnoses. Anti-RgpA was also associated with RA (OR 4.09; 95% CI 1.2–13.9). The combination of anti-RgpA/anti-PPAD showed a high specificity of 93.7% and 82.5% PPV in identifying individuals with RA. RgpA antibodies were associated with the periodontal inflammatory index in RA individuals (p < 0.05). The double positivity of the anti-RgpA/anti-PPAD antibodies enhanced the diagnosis of RA. Therefore, RgpA antibodies and anti-RgpA/anti-PPAD may be biomarkers for R

Antibiotic Susceptibility and Resistance Genes in Oral Clinical Isolates of Prevotella intermedia, Prevotella nigrescens, and Prevotella melaninogenica

The Prevotella genus is a normal constituent of the oral microbiota, and is commonly isolated from mechanically treated polymicrobial infections. However, antibiotic treatment is necessary for some patients. This study compared the antibiotic susceptibility and the presence of resistance genes in clinical oral isolates of P. intermedia, P. nigrescens, and P. melaninogenica. Antibiotic susceptibility was assessed using the agar dilution method. PCR confirmed the species and resistance gene frequency in the Prevotella species. The frequencies of species P. intermedia, P. nigrescens, and P. melaninogenica were 30.2%, 45.7%, and 24.1%, respectively. No isolates of P. intermedia were resistant to amoxicillin/clavulanic acid, tetracycline, or clindamycin. P. nigrescens and P. melaninogenica were resistant to amoxicillin/clavulanic acid and tetracycline at frequencies of 40% and 20%, respectively. P. intermedia was resistant to metronidazole at a frequency of 30%, P. nigrescens at 20%, and P. melaninogenica at 40%. P. nigrescens and P. melaninogenica were resistant to 50% and 10% clindamycin, respectively. The gene most frequently detected was tetQ, at 43.3%, followed by tetM at 36.6%, blaTEM at 26.6%, ermF at 20%, cfxA, cfxA2, and nimAB at 16.6%, and nimAEFI at 3.3%. P. nigrescens was the species with the highest resistance to antibiotics such as amoxicillin/clavulanic acid, amoxicillin, and clindamycin, in addition to being the species with the largest number of genes compared to P. intermedia and P. melaninogenica

Viability and effects on bacterial proteins by oral rinses with hypochlorous acid as active ingredient

This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients

Colombia y Uruguay ante la COVID-19: datos epidemiológicos, medidas generales y en la práctica odontológica

Objective: Analyze the epidemiological data, general and dental practice measures taken by Colombia and Uruguay in the face of the COVID-19 pandemic. Methods: This study is a review. We search in the databases “PubMed”, “Scielo” and in the search engine “Google Scholar”.  Also, were consulted the free databases like google, our world in data and the official pages of the World Health Organization, Ministry of Health of Colombia, The National institute of health and the Ministry of public Health of Uruguay. A descriptive analysis of the epidemiological behavior and the measures taken of dental practice in both countries was done. Results: Colombia had more than 3,000 new cases and more than 100 deaths daily. The opposite occurs in Uruguay, which has managed to decrease the infection and death rates, presenting less than 10 cases and no daily deaths. The struggles experienced by health professionals has increased, dentists are exposed, by their proximity at the time of care, to contact with saliva, blood and the generation of aerosols, therefore, they have modified the biosafety protocols, seeking to prevent infections associated with the practice. Conclusions: Uruguay has an exemplary behavior; in Colombia these results are worrying with an exponential growth. In both countries, dental practice has been affected and new protocols have been implemented that seek to protect the professionals and patients.Objetivo: analizar los datos epidemiológicos y las medidas generales en la práctica odontológica tomadas por Colombia y Uruguay durante la pandemia por la COVID-19.  Métodos: este trabajo es una revisión de la literatura. Se consultaron las bases de datos “PubMed”, “Scielo” y el motor de búsqueda “Google Scholar”. También se consultaron bases de datos de libre acceso como google, our world in data y las páginas oficiales de la Organización Mundial de la Salud, el Ministerio de Salud de Colombia, el Instituto Nacional de Salud y el Ministerio de Salud Pública de Uruguay. Se realizó un análisis descriptivo del comportamiento epidemiológico y de las medidas tomadas en la práctica odontológica en los dos países. Resultados: en Colombia, a diario, se presentan más de 3.000 casos nuevos y más de 100 muertes. Lo contrario ocurre en Uruguay, que ha logrado disminuir las tasas de contagio y de muertes, presentando menos de 10 casos y ninguna muerte diaria. Los profesionales de la salud han incrementado su lucha; los odontólogos se encuentran expuestos por su proximidad al momento de la atención, en el contacto con saliva, sangre y la generación de aerosoles, por lo que han modificado los protocolos de bioseguridad buscando prevenir los contagios asociados a la prestación de servicio. Conclusiones: Uruguay ha tenido un comportamiento ejemplar ante la pandemia; en Colombia los resultados son preocupantes dado el crecimiento exponencial. En ambos países la práctica odontológica se ha visto afectada y se han implementado nuevos protocolos para proteger a profesionales y pacientes

Viability and Effects on Bacterial Proteins by Oral Rinses with Hypochlorous Acid as Active Ingredient

Abstract: This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients

Memorias : XX Congreso Institucional de Investigaciones

Este documento, recoge 10 resúmenes de los trabajos presentados como resultado del proyecto en investigación sobre salud oral e investigación en ingeniería, salud y medio ambiente y biología, para el XX congreso institucional de investigaciones de la Universidad del Bosque. Los productos obtenidos como parte de la investigación son: (1) Condición periodontal en pacientes colombianos con artritis idiopática juvenil (AIJ); (2) Efecto del ácido hipocloroso sobre el sistema amortiguador del pH de la saliva. Estudio in vitro e in vivo; (3) Efecto oxidativo del ácido hipocloroso sobre las proteínas salivales: estudio in vitro; (4) Evaluación del ácido hipocloroso como agente antiplaca para uso en la cavidad oral. Parte I: sustantividad, efecto antiplaca y efectos adversos; (5) Identificación de los factores de virulencia de Klebsiella pneumoniae aislada de cavidad oral asociada a fuentes de contaminación exógena; (6) Modulación de la expresión de factores de crecimiento por ácido hipocloroso sobre fibroblastos gingivales humanos; (7) Potencial de diferenciación y expansión neuronal in vitro a partir de Células Troncales de pulpa dental humana; (8) Resolvina D1 inhibe la expresión de moléculas de adhesión en células endoteliales de arteria coronaria humana estimuladas con lipopolisacárido de P. gingivalis. Estudio in vitro; (9) Viabilidad de microorganismos periodontopáticos y bacilos entéricos después del tratamiento con ácido hipocloroso en comparación con clorhexidina; (10) Modelo in vivo de regeneración ósea mandibular a partir de células troncales dentales humanas.Departamento Administrativo de Ciencia, Tecnología e Innovación [CO] Colciencias1308-519-28960Inducción de disfunción endotelial in vitro por lipopolisacarido de bacterias periodontopaticas e inhibición de la inflamación por resolvina (rvd1) y estatina (rosuvastatina)n