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Administracion sistemica adjunta de moxifloxacina versus ciprofloxacina mas metronidazol en el tratamiento de periodontitis cronica con presencia de bacilos entericos Gram negativos: I. Efectos clinicos y microbiologicos.

ADJUNCTIVE SYSTEMIC ADMINISTRATION OF MOXIFLOXACIN VERSUS CIPROFLOXACIN PLUS METRONIDAZOLE IN THE TREATMENT OF CHRONIC PERIODONTITIS HARBORING GRAM-NEGATIVE ENTERIC RODS: I. MICROBIOLOGICAL AND CLINICAL EFFECTS [1]

INTRODUCCION

Tradicionalmente se ha concluido que el raspaje y alisado radicular (RAR) es un enfoque de tratamiento efectivo, sin embargo, este procedimiento frecuentemente no conduce a los cambios microbiologicos necesarios para mantener a largo plazo la estabilidad de los beneficios clinicos logrados inicialmente. (1)

La utilizacion adjunta de antibioticos administrados sistemicamente ha proporcionado un mejor resultado clinico, particularmente en terminos de reduccion de la profundidad de sondaje (PS) y ganancia del nivel de insercion clinica que el RAR en sujetos con periodontitis cronica. (2, 3) En periodontitis cronica, los patogenos periodontales tales como Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia y Treponema denticola se reducen mas efectivamente empleando antibioticos sistemicos adjuntos. (1, 4-6) Adicionalmente, la terapia antimicrobiana adjunta con antibioticos sistemicos afecta los patogenos periodontales residentes en superficies mucosas no periodontales. (7)

Por otra parte, los bacilos entericos Gram negativos (BEGN) son patogenos oportunistas en un amplio rango de infecciones humanas8 y pueden detectarse en ambientes subgingivales de sujetos con periodontitis. (9-20) Estos microorganismos producen factores de virulencia y tienen capacidad para invadir los tejidos humanos. (11) Los BEGN degradan la elastina y producen enzimas hidroliticas en concurrencia con actividades proteoliticas expresadas por periodontopatogenos reconocidos, durante la destruccion de los tejidos de soporte de los dientes. (11) En los sitios periodontales el aislamiento de elastina degradada por cepas de Klebsiella pneumonia, un BEGN usual que puede causar neumonia, enfatiza que la cavidad oral es un reservorio de patogenos que facilita la diseminacion, causando infecciones severas en el tracto respiratorio bajo. (11)

La lecitinasa producida por los BEGN en sitios periodontales puede alterar las vias de transduccion en las celulas endoteliales, plaquetas y neutrofilos, conduciendo a una produccion descontrolada de mediadores intracelulares y moleculas de adhesion, alterando asi el trafico de granulocitos al tejido infectado. (11)

Los BEGN subgingivales persisten a menudo despues de desbridamientos y cirugia, y se ha implicado como patogeno clave en casos de periodontitis refractaria. (10, 12-15) Estos microorganismos se detectaron en alta frecuencia y elevadas proporciones en pacientes con fracasos de implantes. (13) Adicionalmente, han mostrado baja sensibilidad a la clorhexidina (16) y el hecho que estos microorganismos muestren resistencia in vitro a la mayoria de antibioticos adjuntos usados para tratar la periodontitis, (17-20) indica que las lesiones periodontales asociadas con estos microorganismos, no responden a modalidades de tratamiento convencional. (10) La terapia con ciprofloxacina tiene el potencial de erradicar los BEGN de las bolsas periodontales, pero tiene poca actividad contra los principales patogenos periodontales, (10, 21) por lo tanto, debe combinarse con un agente antimicrobiano activo contra anaerobios como el metronidazol. (9, 10) Por el contrario, en publicaciones previas, la moxifloxacina (MOX) ha demostrado actividad in vitro contra los BEGN, (20) e in vitro (22) y eficacia in vivo (4) contra periodontopatogenos. Estas publicaciones tambien han recomendado la realizacion de ensayos clinicos que investiguen el efecto de la MOX en el tratamiento de pacientes con periodontitis cronica que presentan BEGN en placa subgingival.

No se conocen estudios que hayan evaluado la eficacia in vivo y la aplicabilidad clinica de la MOX contra BEGN presentes en placa subgingival. Por lo tanto, el objetivo del presente articulo fue evaluar y comparar los efectos clinicos y microbiologicos del RAR combinado con aplicacion sistemica de MOX o ciprofloxacina mas metronidazol (CIPRO + MET) en el tratamiento de pacientes con periodontitis cronica.

MATERIALES Y METODOS

Sujetos

Se reclutaron 76 pacientes sistemicamente saludables (45 mujeres y 31 hombres), con edades entre los 27 y 66 anos, que asistieron a las clinicas odontologicas de la Universidad de Antioquia, entre octubre de 2008 y marzo de 2009. Cada participante firmo un consentimiento.

El diseno del estudio fue aprobado por el Comite de Etica de la Sede de Investigacion Universitaria de la Universidad de Antioquia de acuerdo con la declaracion de Helsinki sobre experimentacion en humanos. Se consideraron candidatos para este estudio, pacientes con diagnostico de periodontitis cronica. Los pacientes fueron mayores de 26 anos, con al menos 20 dientes naturales, incluyendo un molar en cada cuadrante y con por lo menos ocho sitios con PS [mayor que o igual a] 5 mm. Los criterios de exclusion circunscribian pacientes diabeticos, enfermedades cardiovasculares o cualquier enfermedad sistemica que pudiera alterar el curso de la periodontitis. Tambien fueron criterios de exclusion mujeres embarazadas o lactantes, consumo de antimicrobianos o antiinflamatorios en los ultimos seis meses, y terapia periodontal en el ultimo semestre.

Diseno experimental y tratamiento

Este ensayo clinico controlado de brazos paralelos con enmascaramiento del examinador, clinico y estadistico, tuvo seis meses de seguimiento. Los sujetos se asignaron aleatoriamente para recibir uno de los tratamientos, mediante una tabla generada por computador. La asignacion de los pacientes al grupo de tratamiento, se llevo a cabo por un coordinador de clinica independiente del grupo de investigacion. El codigo de aleatorizacion se mantuvo oculto por este coordinador y no se rompio hasta despues del analisis de los datos. Los dos grupos de tratamiento consistieron en RAR combinado con MOX aplicado sistemicamente en una dosis de 400 mg diarios por siete dias (grupo MOX) o RAR combinado sistemicamente con CIPRO + MET en una dosis de 1 g diario mas 500 mg dos veces al dia por 7 dias (grupo CIPRO + MET). Bajo anestesia local, un periodoncista experimentado hizo un RAR boca completa durante aproximadamente dos horas y media. El objetivo final del RAR fue una superficie radicular lisa al tacto. La ingestion de los antimicrobianos adjuntos se inicio en la visita de RAR. Los sujetos del grupo MOX y CIPRO + MET se informaron extensamente acerca de la importancia de la ingestion de la medicacion prescrita.

Los pacientes se monitorearon clinica y microbiologicamente en la linea de base (antes de la terapia) y a los 3 y 6 meses posterapia. Durante estas sesiones, se evaluo la higiene oral y se reenfatizo en los cuidados orales caseros. Todos los sujetos asistieron a las citas de revision y recibieron evaluaciones de higiene oral.

Las citas de revision se hicieron cada dos semanas durante los seis meses despues del tratamiento.

Cumplimiento

Un asistente dental llamo por telefono a cada sujeto durante los siguientes 6 dias para recordarle la ingestion de las dosis restantes. El mismo asistente dental, que no participo en el proceso de aleatorizacion, registro el cumplimiento de la ingesta de medicamentos y la aparicion de eventos adversos. A los sujetos se les pidio devolver las cajas con los medicamentos la semana despues de la visita de RAR con el fin de comprobar cualquier inexactitud en el consumo de los antibioticos.

Evaluacion clinica

A cada paciente se le hizo una historia medica y se le practicaron examenes clinicos y radiograficos. En cada visita de monitoreo se observo placa visible (0/1), sangrado al sondaje (SS) (0/1), profundidad de sondaje (PS) (en mm) y nivel de insercion clinica (NIC) (en mm) en seis sitios por diente (mesobucal, bucal, distobucal, distolingual, lingual y mesolingual) en todos los dientes, excluyendo el tercer molar. Las medidas PS y NIC se registraron al milimetro mas cercano, utilizando una sonda periodontal calibrada (UNC-15, Hu-Friedy, Chicago, IL). Las mediciones de los pacientes en todas las visitas, se hicieron por el mismo clinico entrenado, cegado y calibrado. El periodoncista que efectuo los examenes clinicos no hizo la terapia en los pacientes. La reproducibilidad intraexaminador se evaluo antes y durante el periodo experimental, de acuerdo con el metodo descrito por Araujo et al. (23) Se hicieron medidas repetidas en un total de 10 pacientes periodontales (no participantes en el estudio), cinco de los cuales se examinaron antes del ensayo clinico y los otros cinco durante la fase experimental. Se condujeron medidas por duplicado en cada paciente con al menos dos horas entre cada examen. El coeficiente de correlacion intraclase para el promedio de PS y NIC fue 0,92 y 0,91, respectivamente. La reproducibilidad de los examinadores fue similar antes y durante el estudio. El diagnostico de periodontitis cronica se baso en los criterios definidos por la Academia Americana de Periodoncia (AAP). (24)

Muestreo microbiologico

Se tomaron muestras microbiologicas de los pacientes con periodontitis en sitios con profundidad de sondaje [mayor que o igual a] 5 mm. Para la toma de muestras se seleccionaron las seis bolsas periodontales mas profundas de cada paciente. Despues de aislar la zona con algodon y eliminar la placa supragingival con cureta, se insertaron puntas de papel esteril (Maillefer, Ballaigues, Suiza) en cada bolsa periodontal durante 20 s. Las muestras de cada paciente se depositaron en 2 ml de medio de transporte (Viability Medium Goteborg Anaerobically III: VMGA III) (25) y se llevaron al laboratorio de microbiologia de la Facultad de Odontologia de la Universidad de Antioquia para procesarlas dentro de las dos horas siguientes. Las muestras fueron analizadas utilizando tecnicas de cultivo para la presencia de periodontopatogenos de acuerdo con Slots. (26) Todas las muestras fueron procesadas antes de 24 horas a temperatura ambiente e incubadas inmediatamente en C[O.sub.2] y sistemas de cultivo anaerobicos. Las cajas de agar Brucella se incubaron a 35[grados]C en anaerobiosis por siete dias. Se incubo tripticasa soya con suero, bacitracina y vancomicina en 10% de C[O.sub.2] en aire a 37[grados]C por 4 dias. Se hizo identificacion presuntiva de acuerdo con metodos descritos (26, 27) y usando sistemas de identificacion comercial (RapID ANA, Remel, Norcross, GA) para A. actinomycetemcomitans, P. gingivalis y T. forsythia. El conteo viable total (CVT) se definio como el numero total de unidades formadoras de colonias obtenidas sobre medios no selectivos. Se enumeraron las especies encontradas sobre medios selectivos y se calculo su porcentaje de CVT.

Aislamiento de BEGN por cultivo. Despues de 20 s, las puntas de papel se almacenaron en 2,0 ml de VMGA III. (25) Las muestras se mantuvieron a temperatura ambiente, transferidas al laboratorio y procesadas 4 h despues del muestreo. Despues de colocar los viales en una incubadora por 30 min a 37[grados]C, la placa bacteriana se disperso mecanicamente con un tubo mezclador en ajuste maximo por 60 s. Se prepararon 10 diluciones en serie en agua pepton, y las alicuotas se colocaron en agar MacConkey. Las placas se incubaron aerobicamente a 37[grados]C por 24 h. Cada aislamiento se caracterizo de acuerdo con la colonia y morfologia celular y caracteristicas de pigmentacion Gram. Los BEGN se clasificaron usando una prueba bioquimica estandar (BD, Sparks, MD). CVT se definio como el numero total de unidades formadoras de colonias obtenidas sobre medios no selectivos. Se enumeraron las especies encontradas sobre medios selectivos y se calculo su porcentaje de CVT.

Cada paciente proporciono una muestra de placa subgingival. Se utilizo igual numero de aislamientos de cada paciente.

Analisis estadistico

Los datos se introdujeron en una base de datos Excel (Microsoft Office 2007) y se verificaron errores de digitacion. El sujeto fue la unidad de analisis. Para la descripcion de los datos se utilizaron promedios [+ o -] desviacion estandar, y proporciones de sitios dentro de categorias. Se utilizo la prueba Kolmogorov-Smirnov para verificar la distribucion normal de las variables continuas. Los datos categoricos se analizaron con pruebas de Chi cuadrado, y las comparaciones entre los dos grupos se hicieron con la prueba Mann-Whitney. Las diferencias entre los grupos y entre los momentos de evaluacion al interior de cada grupo se calcularon mediante pruebas de Mann-Whitney y Wilcoxon, respectivamente.

Se incluyeron en el analisis estadistico solamente los sitios con una PS [mayor que o igual a] 4 mm, debido a que los sitios menos profundos no recibieron RAR, aun cuando se trataron con detartraje subgingival. En este estudio se establecio PS como resultado primario y NIC como secundario. Se registraron como variables explicativas el SS y la presencia de entericos y de periodontopatogenos evaluados.

Se analizaron separadamente los datos relacionados con PS > 6 mm, empleando el sitio como unidad observacional. Se calculo la proporcion de sitios > 6 mm en cada momento y para cada grupo y se compararon los cambios de estas proporciones desde la linea de base entre los grupos usando la prueba Mann-Whitney.

Los datos microbiologicos se calcularon teniendo en cuenta al sujeto como unidad de analisis. En todas las evaluaciones se estimo el numero de sujetos positivos para las especies investigadas en cualquiera de los sitios muestreados. Se aplico la prueba Mann-Whitney con el fin de identificar las diferencias especificas, mientras que las diferencias entre cada grupo se evaluo con la prueba Wilcoxon. Se determino 0,05 como el nivel de significancia estadistica para todas las pruebas. Para el manejo de todas las pruebas estadisticas se utilizo el mismo paquete estadistico (SPSS, Statistical Package for the Social Sciences, version 15, Chicago, IL).

Calculo del tamano de muestra

El calculo del tamano de muestra se baso en el sujeto como unidad de analisis para estudios aleatorizados. Con el fin de calcular el tamano ideal de muestra que asegurara un adecuado poder en este ensayo clinico, se consideraron diferencias de al menos 1 mm de NIC y desviacion estandar de 1,1 mm entre los grupos en bolsas periodontales inicialmente profundas (> 6 mm). (6) Segun este calculo, se determino que una muestra [mayor que o igual a] 19 sujetos por grupo seria necesario para proporcionar el 80% de poder con un [alfa] de 0,05.

RESULTADOS

Retencion de sujetos

98 sujetos se evaluaron para su elegibilidad antes de ingresar al estudio. De este grupo, 22 individuos fueron excluidos debido a que no cumplieron los criterios de inclusion. De los 76 pacientes reclutados, 72 completaron los datos para las tres visitas de monitoreo, mientras que dos sujetos no asistieron a una cita. En los dos sujetos con datos perdidos se hizo un analisis por intencion de tratar, en los que la ultima observacion se llevo hacia adelante, y quedaron en total 74 individuos con datos completos que fueron incluidos en el analisis. Los dos pacientes restantes (uno de cada grupo) tuvieron datos de base solamente y no fueron incluidos en el analisis. La participacion de los individuos durante el estudio se ilustra en la figura 1.

[FIGURA 1 OMITIR]

Eventos adversos

Todos los sujetos que terminaron el estudio Informaron completa adherencia al esquema de antibioticos prescrito. Siete sujetos del grupo CIPRO + MET manifestaron eventos adversos durante el estudio, tales como diarrea y nauseas. Ademas, tres pacientes del mismo grupo reportaron mareo. La mayoria de pacientes del grupo MOX afirmaron que la medicacion no ocasiono mayores disturbios en su rutina diaria.

Hallazgos clinicos

La tabla 1 presenta las caracteristicas clinicas y demograficas de base de los 76 pacientes subdivididos en los dos grupos de tratamiento. Excepto por la edad, no se observaron diferencias estadisticas entre los grupos de tratamiento.

La tabla 2 muestra la estadistica descriptiva y las comparaciones para ambos grupos relacionadas con SS, PS y NIC. No se observo diferencias significativas entre ellos durante ninguna evaluacion. Cuando se hicieron comparaciones en cada grupo, ambos mostraron una reduccion significativa en PS y SS comparado con la linea de base (P < 0,001), y esta diferencia se mantuvo en los 6 meses desde la linea de base en los dos grupos. No se observo ninguna diferencia en los grupos a los 3 y 6 meses. Cuando se hicieron comparaciones en cada grupo, los dos presentaron mejoria significativa en el NIC comparado con la linea de base (P < 0,001), y esta diferencia se mantuvo a los 6 meses en ambos. De la misma forma, no se observaron diferencias en los grupos a los 3 y 6 meses.

Se buscaron diferencias adicionales analizando bolsas con PS > 6 mm (tabla 3) y utilizando el sitio en vez del sujeto como unidad observacional, considerando el efecto de los tratamientos sobre el resultado primario. De acuerdo con los hallazgos, a los seis meses los sujetos en los dos grupos, mostraron la mayor reduccion desde la linea de base en proporciones de sitios con PS > 6 mm (P < 0,001). Ninguna diferencia se observo entre los individuos que recibieron MOX o CIPRO + MET.

Hallazgos microbiologicos

En la tabla 4 se presenta el numero de sujetos positivos a las especies investigadas. No se observaron diferencias significativas entre los grupos en la linea de base. Igualmente, no se observo ninguna diferencia entre los grupos en ningun momento. En los sujetos, la administracion de los antimicrobianos condujo a la eliminacion y reduccion de la mayoria de las bacterias, tres meses despues del inicio del estudio. Tres meses despues de la linea de base no se identificaron BEGN ni A. actinomycetemcomitans en ambos grupos, y tambien se redujeron significativamente P. gingivalis y T. forsythia (P < 0,05). A los seis meses, se observo recolonizacion de las especies investigadas, excepto para los BEGN y A. actinomycetemcomitans. Sin embargo, P. gingivalis y T. forsythia se disminuyeron significativamente desde la linea de base (P < 0,05).

DISCUSION

Este ensayo clinico aleatorizado evaluo los efectos clinicos y microbiologicos del RAR en combinacion con antibioticos adjuntos (MOX versus CIPRO + MET) en el tratamiento de sujetos con periodontitis cronica asociada a BEGN. Estas bacterias se observan con frecuencia en la microbiota subgingival de pacientes considerados clinicamente refractarios a la terapia periodontal convencional mecanica y antibiotica. (12-15) El alto potencial patogenico de los BEGN, (10) junto con las altas proporciones de lesiones refractarias, (10, 12-15) los involucra en la patogenesis de algunas formas de periodotitis. (17)

Esto sugiere la necesidad de una antibioterapia apropiada para el tratamiento de infecciones periodontales que incluyen BEGN. (10, 12-15) Ademas, los BEGN pueden ser patogenos claves en infecciones severas del tracto urinario, piel, hueso y articulaciones, y varios sitios del cuerpo (8, 11, 28) elaborando un numero de potentes enzimas y toxinas de posible relevancia para la destruccion de los tejidos periodontales. (11)

Se selecciono la utilizacion adjunta de MOX o ciprofloxacina con base en resultados preliminares in vitro (7, 17-21) e in vivo (4, 10) que sugirieron beneficios clinicos y microbiologicos en el tratamiento de periodontitis asociadas a BEGN. Para nuestro conocimiento, el presente estudio es el primer ensayo clinico que compara MOX y CIPRO + MET como terapia adjunta al RAR.

Datos clinicos

Las dos terapias usadas en el presente estudio mejoraron los parametros clinicos evaluados. El RAR combinado con MOX o CIPRO + MET fue igualmente efectivo en mejorar PS y NIC. El mismo patron se observo para el parametro SS y no se observaron diferencias significativas en los sujetos entre los grupos en ningun momento. Para nuestro conocimiento, hasta la fecha solamente dos estudios han investigado independientemente la eficacia de la moxifloxacina adjunta (4) y de la ciprofloxacina (10) en periodontitis. De acuerdo con esto, nuestros resultados concuerdan con los de Guentsch y colaboradores, (4) mostrando cuando MOX se combino con RAR en el tratamiento de periodontitis cronica presento mejor respuesta clinica. Por otra parte, es importante notar que los pacientes incluidos en el estudio con ciprofloxacina (10) tuvieron historia de periodontitis refractaria asociada a BEGN. Este hallazgo es especialmente interesante debido a que la terapia mecanica sola fue incapaz de suprimir completamente estos organismos en lesiones con periodontitis. (10) En ese estudio y similar a la presente investigacion, la terapia con ciprofloxacina sistemica adjunta demostro mejoria en SS, PS y NIC. Los resultados de la presente investigacion concuerdan con estudios que sugieren que el metronidazol sistemico solo o combinado mejora significativamente los resultados clinicos del desbridamiento periodontal no quirurgico. (1-3, 6) Es interesante mencionar que Cionca y colaboradores,1 demostraron recientemente que la adicion de agentes antimicrobianos al tratamiento mecanico puede reducir la necesidad de terapia posterior.

Cuando se analizan los datos en el sitio, y en particular la proporcion de sitios con PS > 6 mm, se observo entre los grupos patron similar. El grupo MOX y el grupo CIPRO + MET exhibieron reduccion significativa de los porcentajes de sitios > 6 mm. Este hallazgo indica un efecto benefico de estos antimicrobianos en bolsas profundas. Las conclusiones del presente estudio concuerdan con un ensayo clinico recientemente publicado por Guentsch y colaboradores. (4) Estos autores tambien demostraron que la terapia sistemica ajunta de MOX por 7 dias produce reduccion adicional de la PS en bolsas profundas (> 6 mm) a los 6 meses en periodontitis cronica. Otro estudio demostro resultados similares con ciprofloxacina, despues de 12 meses, en pacientes con periodontitis refractaria y BEGN. (10) Dos revisiones sistematicas que evaluaron el efecto de antibioticos administrados sistemicamente sugieren que los antimicrobianos proporcionan mayor beneficio en sujetos con mas enfermedad periodontal y en sitios periodontales mas profundos. (2, 3)

Datos microbiologicos

Este es el primer ensayo clinico que hasta la fecha ha evaluado los cambios microbiologicos que ocurren con el uso de CIPRO + MET y el segundo que evalua MOX en el tratamiento de la enfermedad periodontal, y el primero que compara estos dos protocolos de tratamiento. Nos enfocamos en la evaluacion de BEGN y tres periodontopatogenos (A. actinomycetemcomitans, P. gingivalis y T. forsythia). Las razones para estudiar BEGN se explicaron previamente. Por otra parte, se ha demostrado que una disminucion en el recuento de los periodontopatogenos estudiados en esta investigacion se asocio con mejoria clinica; estos microorganismos ademas son predictores de los resultados terapeuticos. (5)

Nuestra publicacion previa20 reporto cuatro especies de BEGN en placa subgingival en 20 (26.31%) de 76 pacientes: K. pneumoniae occurio en 12 pacientes, Pseudomonas aeruginosa en cuatro sujetos y se identificaron otras tres especies con baja prevalencia. En Latinoamerica su ocurrencia varia ampliamente dentro de la poblacion estudiada, alcanzando una prevalencia de 17,6% en Chile (29) y 67% en Santo Domingo. (30) Entre brasilenos (18) y colombianos (19, 29) se reportaron frecuencias similares a las encontradas en nuestro estudio. (20)

Igualmente, en esta investigacion, la presencia de periodontopatogenos observada fue similar a la encontrada por investigaciones previas. (19, 29)

Es importante notar que las colonias de BEGN son de mayor tamano (19) y que estos organismos pueden colonizar las bolsas periodontales en altas proporciones. Por otra parte la reaccion en cadena de la polimerasa (RCP) no tiene en consideracion si la muestra es viable, favoreciendo la mayor frecuencia. (19) D'Ercole y colaboradores31 compararon recientemente metodos de cultivo convencional y multiplex RCP para la deteccion de periodontopatogenos, y encontraron que para los dos metodos hubo un buen acuerdo de exactitud para la determinacion de A. actinomycetemcomitans y P. gingivalis. Resultados similares se obtuvieron para T. forsythia. (32) Las tecnicas de cultivo y RCP introducen muchos problemas metodologicos cuando se aplican en microbiologia oral; sin embargo, no se ha desarrollado aun la tecnica ideal para la deteccion de patogenos en muestras de placa subgingival. (31) Al igual que Botero y colaboradores, (19) el presente estudio reporta la frecuencia de estos microorganismos basados en tecnicas de cultivo debido a que permite trabajar posteriormente con los mismos. Se requieren posteriores estudios que clarifiquen el papel de los BEGN sobre los parametros clinicos y la respuesta al tratamiento periodontal.

De acuerdo con los resultados clinicos, los dos grupos presentaron cambios favorables en el perfil microbiologico subgingival despues del tratamiento. Esta observacion es consistente con diferentes estudios, (1, 4, 6) en los que se asocio significativamente una disminucion de la ocurrencia de las especies evaluadas con mejoria en el NIC y disminucion en la PS. La mayor disminucion de la prevalencia se presento entre la linea de base y el muestreo a los tres meses. Estos resultados estan de acuerdo con otros estudios que tambien demostraron el efecto adjunto de estos antibioticos para reducir las especies del complejo rojo (1, 4-6) y los BEGN. (10) Es de interes aportar que en el presente estudio, ambos protocolos de tratamiento redujeron significativamente los BEGN y el A. actinomycetemcomitans a niveles indetectables, despues de tres y seis meses. Esta observacion es consistente con los hallazgos descritos por Slots y colaboradores.10 y Guentsch y colaboradores, (4) quienes encontraron que los BEGN y el A. actinomycetemcomitans se redujeron a niveles indetectables despues de usar ciprofloxacina adjunta y MOX respectivamente. Como se observo en estudios previos, (1, 4-6) para P. gingivalis y T. forsythia se detecto un leve incremento despues de tres meses.

Los sujetos tratados en la presente investigacion mostraron buena respuesta clinica, sugiriendo que la disminucion en el recuento de periodontopatogenos y BEGN puede ser crucial para una terapia periodontal exitosa y estabilidad a largo plazo. (5, 10) El seguimiento longitudinal de estos sujetos ayudara a clarificar esta hipotesis.

Los BEGN y periodontopatogenos investigados fueron altamente sensibles a las fluoro-quinolonas ciprofloxacina y moxifloxacina. (20, 22) Las concentraciones de ciprofloxacina observadas en el fluido crevicular son aproximadamente de 2,5 [micron]g/ml y son varias veces mas elevadas que los niveles encontrados normalmente en el plasma. (33) Esto puede deberse al incremento de las quinolonas en los granulocitos polimorfonucleares. (33) En dosis terapeuticas, el medicamento aparece en la saliva a los mismos niveles logrados en el suero. (33) Su baja actividad contra anaerobios Gram negativos limita frecuentemente su uso en periodoncia a combinaciones con metronidazol. (10) En contraste, la nueva 8-methoxy quinolona moxifloxacina tiene efectiva accion antimicrobiana contra anaerobios Gram negativos y Gram positivos. (20, 22) Muller y colaboradores.34 demostraron que todas las cepas de A. actinomycetemcomitans fueron sensibles a moxifloxacina a 0,032 [micron]g/ml. Las concentraciones en saliva y plasma reflejan estrechamente las concentraciones equivalentes en el plasma venoso, sin embargo sobrepasan los niveles plasmaticos. (33) Las quinolonas son reconocidas por su modulacion en la respuesta inmune, permitiendo la exterminacion del A. actinomycetemcomitans por parte de los leucocitos polimorfonucleares. (35)

Eventos adversos

Diez sujetos (26,3%) del grupo CIPRO + MET reportaron eventos adversos durante el estudio. Estos resultados estan de acuerdo con una revision sistematica2 que evaluo los efectos de los antibioticos administrados sistemicamente, sugiriendo que el 39% de los sujetos en el grupo experimental presentaron diarrea cuando se indicaba metronidazol solo o combinado. Ademas, la ciprofloxacina se ha asociado con raros pero importantes eventos adversos clinicos en pacientes con dano en la funcion renal (36) y en los Estados Unidos se han reportado varios casos de "Torsades de Pointes". (37) Por el contrario, los sujetos del grupo MOX no reportaron eventos adversos durante esta investigacion, corroborando los resultados de Guentsch y colaboradores. (4)

Ademas de las actividades antimicrobianas, tambien se han estudiado las propiedades de la moxifloxacina, que tiene excelente biocompatibilidad, larga vida media y buena penetracion tisular. (38) Igualmente, tiene excelente tolerancia. (4) Sus propiedades farmacocineticas permiten una solo dosis diaria. Esto reduce los costos y aumenta la adherencia del paciente. (39) Este es un factor importante, debido a que una incompleta adherencia a una prescripcion antibiotica sistemica por 7 dias se asocio con la disminucion de los resultados clinicos en pacientes con periodontitis. (40)

El presente estudio tuvo limitaciones. Fue un estudio aleatorizado, pero no controlado con placebo. Se considero suministrar un placebo pero los diversos regimenes de dosis y la variedad de presentacion de los antibioticos hacian muy complejo obtener un placebo estandar. Por lo tanto se decidio no usar placebo. Sin embargo, el examinador no fue consciente del grupo de tratamiento al cual se asignaron los sujetos. Situaciones similares se les presentaron a Haffajee y colaboradores (6) y Guentsch y colaboradores (4) en dos ensayos clinicos recientemente publicados. En el presente estudio no se utilizo otro grupo que incluyera solamente tratamiento mecanico. Distinguidos investigadores han recomendado la administracion sistemica de antibioticos en pacientes con periodontitis cronica que presentan BEGN. (10, 12-15) Ademas, dos revisiones sistematicas han demostrado que el uso adjunto de antibioticos administrados sistemicamente proporcionan mejor resultado clinico, particularmente en terminos de reduccion de PS y ganancia de nivel de insercion clinica, que el RAR, no solamente en formas agresivas de periodontitis si no tambien en pacientes con periodontitis cronica. (2, 3) Finalmente, se reporto recientemente que la adicion de agentes antimicrobianos a la terapia mecanica puede reducir la necesidad de un tratamiento posterior. (1) Por lo tanto, en el presente protocolo no se utilizo un grupo de solo tratamiento mecanico. A pesar de sus limitaciones, la decision de incorporar MOX o CIPRO-MET adjunto al tratamiento mecanico se confirma por los resultados de la presente investigacion.

Los actuales protocolos para el tratamiento antibiotico periodontal se basan en estudios microbiologicos hechos fuera de Latinoamerica.

Consecuentemente la prevalencia de diferencias considerables en la composicion de la flora subgingival en diversas poblaciones puede incrementar la posibilidad de desarrollar resistencia antibiotica o promover un sobrecrecimiento de organismos superinfectantes. (41) La moxifloxacina ofrece diferentes ventajas sobre las fluoroquinolonas previamente disponibles incluyendo reducida posibilidad para promover el desarrollo de resistencia.11 Aun cuando los datos publicados y la experiencia clinica con estos agentes es limitada, dado su reciente ingreso a nuestro mercado, esta perspectiva intenta proporcionar un entendimiento del papel potencial de la moxifloxacina en el tratamiento de pacientes con periodontitis cronica que presentan bacilos entericos en placa subgingival.

CONCLUSIONES

Entre las limitaciones de este estudio, los resultados indican que los pacientes con periodontitis cronica que presentan bacilos entericos Gram negativos en placa subgingival pueden beneficiarse de la administracion sistemica de la moxifloxacina o ciprofloxacina mas metronidazol como terapia antimicrobiana adjunta al tratamiento mecanico. Las dos estrategias resultaron en ganancia significativa del nivel de insercion clinica y disminucion de la profundidad de sondaje periodontal. Este mismo patron se observo para el SS. Se demostro la supresion de BEGN por debajo de niveles detectables por al menos 6 meses. Consecuentemente, el uso sistemico de moxifloxacina o ciprofloxacina mas metronidazol adjunto a la terapia convencional puede ser una alternativa para el tratamiento de pacientes con periodontitis cronica. La seleccion del metodo de tratamiento se debe basar en la adherencia y la ausencia de efectos adversos potenciales.

INTRODUCTION

It has been commonly concluded that scaling and root planing (SRP) is an effective treatment approach; however, this procedure does not frequently lead to the microbiological changes necessary for maintaining the long-term stability of the clinical benefits initially achieved. (1)

The adjunctive use of systemically administered antibiotics has been shown to provide a better clinical outcome, particularly in terms of probing depth (PD) reduction and attachment-level gain than SRP in subjects with chronic periodontitis. (2, 3) Periodontal pathogens such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are more effectively reduced by the use of systemic antibiotics in chronic periodontitis. (1, 4-6) Furthermore, adjunctive antimicrobial therapy with systemic antibiotics affects periodontal pathogens residing in non-periodontal mucosal surfaces. (7)

On the other hand, Gram-negative enteric rods (GNER) are opportunistic pathogens in a wide range of human infections (8) and they can be detected in the subgingival environment of periodontitis subjects. (9-20) These microorganisms are able to produce virulence factors and have shown the capacity to invade human tissue. (11) GNER are capable of degrading elastin and have been shown to produce hydrolytic enzymes that might accord with proteolytic activities expressed by recognized periodontopathogens, during destruction of tooth-supporting tissues.11 The isolation from periodontal sites of elastin degrading Klebsiella pneumonia strain, a usual GNER that may cause pneumonia, emphasizes that the oral cavity is a reservoir of pathogens that may spread and cause severe infections in the lower respiratory tract. (11)

Lecithinase production by GNER strains from periodontal lesions may disrupt transduction pathways in endothelial cells, platelets and neutrophils leading to uncontrolled production of intracellular mediators and adhesion molecules, thus altering the traffic of granulocytes to the infected tissue. (11)

Subgingival GNER often persist after periodontal debridement and surgery and have been implicated as key pathogens in cases of refractory periodontitis. (10, 12-15) These microorganisms were detected at higher frequency and in higher proportions in patients with failing implants. (13) Additionally, they show less susceptibility to chlorhexidine (16) and the fact that these microorganisms exhibit in vitro resistance to the majority of adjunctive antibiotics used to treat periodontitis, (17-20) means that periodontal lesions associated with these organisms do not respond to conventional treatment modalities. (10) Ciprofloxacin therapy has the potential to eradicate GNER from periodontal pockets but has little activity against the main putative periodontopathogens, (10, 21) therefore, it should be combined with an antimicrobial agent active against anaerobes as metronidazole. (9,10) Conversely, in earlier papers, moxifloxacin (MOX) has shown in vitro activity against GNER, (20) and in vitro (22) and in vivo (4) efficacy against periodontopathogens. These articles also outlined a procedure for clinical trials to investigate the effects of MOX in the treatment of patients with chronic periodontitis harboring GNER in subgingival plaque.

To our knowledge no studies have assessed the in vivo efficacy and clinical applicability of MOX against GNER present in subgingival plaque. Therefore, the aim of the present study was to evaluate and compare the clinical and microbiological effects of SRP combined with systemic MOX or ciprofloxacin plus metronidazole (CIPRO + MET) in the treatment of subjects with chronic periodontitis.

MATERIALS AND METHODS

Subjects

Seventy-six systemically healthy subjects (45 women and 31 men), aged 27 to 66 years who attended the dental clinics of Universidad de Antioquia were recruited from October 2008 to March 2009. Informed and written consent was obtained from each participant.

The study design was approved by the Ethics Committee on Human Research of the University Investigation Department of the Universidad de Antioquia according to the Declaration of Helsinki on experimentation involving human subjects. Patients with a diagnosis of chronic periodontitis were considered candidates for the study. Subjects were >26 years of age, had at least 20 natural teeth, including at least 1 molar tooth in each quadrant, and at least eight sites with PD [greater than or equal to] 5 mm. Exclusion criteria included diabetes, cardiovascular disease, or any other systemic disease that could alter the course of periodontal disease. Pregnant or nursing women, consumption of systemic antimicrobials or anti-inflammatory drugs in the last six months, and periodontal therapy during the last six months also served as exclusion criteria.

Experimental design and treatment

This was a parallel arm, randomized trial with masking of examiner, clinician performing treatment and statistician, with 6 months of follow-up. Subjects were randomly assigned by a computer-generated table to receive one of the two treatments. The assignment of subjects to the treatment groups was carried out by the clinic coordinator remote from the study. The randomization code was held centrally by the clinic coordinator and was not broken until completion of the data analyses. The two treatment groups consisted of SRP combined with systemically administered MOX at the dosage of 400 mg once daily for 7 days (MOX group) or SRP combined with systemically administered CIPRO+MET at the dosage of 1g once daily plus 500 mg b.i.d. for 7 days (CIPRO + MET group). One-stage full mouth SRP under local anesthesia was performed in approximately two hours and half by the same experienced periodontist. The endpoint of SRP was a tactile smooth root surface. The adjunctive agents were started at the SRP visit. Subjects in the MOX and CIPRO + MET groups were extensively informed about the intake of the prescribed medication.

Subjects were clinically and microbiologically monitored at baseline (before therapy) and at 3 and 6 months post-therapy. During the monitored sessions, oral hygiene was evaluated and home care instructions were re-emphasized. All subjects came for recall visits and received oral hygiene evaluations.

The recall visits were made on a 2-week interval during the 6 months after treatment.

Compliance

A dental assistant called each subject during the next 6 days by telephone to remind him/her to take the remaining doses. The same dental assistant, not involved in the randomization process, recorded compliance with medication intake and occurrence of adverse events. The subjects were asked to bring the boxes containing the medication the week after the SRP visit, when the pills were counted in order to check any inaccuracy in drug taking.

Clinical evaluation

Medical history was taken and clinical and radiographic examinations were conducted for each patient. At each monitoring visit, visible plaque (0/1), bleeding on probing (BOP) (0/1), probing depth (PD) (in mm) and clinical attachment level (CAL) (in mm) were measured at six sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual and mesiolingual) in all teeth excluding third molars. The PD and CAL measurements were recorded to the nearest millimeter by a calibrated standard probe (UNC-15, Hu-Friedy, Chicago, IL). Measurements at all visits for a given subject were made by the same blinded, trained and calibrated clinician. The clinician making the clinical measurements did not perform the therapy on the subjects. The intra-examiner reproducibility was assessed before and during the experimental period according to the method described by Araujo et al. (23) Repeated measurements were performed on a total of 10 periodontal patients (who were not participating in this study), five of whom were examined immediately before the clinical trial, and the other five during the experimental period. Duplicate measurements were conducted in groups of two patients with at least 2 h between each examination. The intra-class correlation coefficients for mean PD and CAL were 0.92 and 0.91, respectively. The examiner's reproducibility of measurements made before and during the study was similar. The diagnosis of chronic periodontitis was made based on criteria defined at the workshop sponsored by the American Academy of Periodontology (AAP). (24)

Microbial sampling

Microbial sampling on periodontitis patients was performed on pockets [greater than or equal to] 5 mm. The deepest six pockets were selected for sampling. After removing supragingival plaque with curets and isolating the area with cotton pellets, the paper points (Maillefer, Ballaigues, Switzerland) were inserted into each periodontal pocket for 20 seconds. The paper points were transferred to a tube with Viability Medium Goteborg Anaerobically (VGMA) III medium.25 The samples were analyzed using microbial culture techniques for the presence of periodontopathic bacteria according to Slots. (26) All samples were processed in [less than or equal to] 24 hours at room temperature and immediately incubated in C[O.sub.2] and anaerobic culture systems. Brucella blood agar medium was incubated at 35[degrees]C in an anaerobic jar for 7 days. The trypticase-soy with serum, bacitracin, and vancomycin medium was incubated in 10% C[O.sub.2] in air at 37[degrees]C for 4 days. Presumptive identification was performed according to the methods described (26, 27) and using a commercial identification micromethod system (RapID ANA, Remel, Norcross, GA) for A. actinomycetemcomitans, P. gingivalis and T. forsythia. Total viable counts (TVC) were defined as the total number of colony-forming units obtained on non-selective media plates. Species found on selective media were enumerated and their percentage of TVC was calculated.

Isolation of GNER by culture. After placement for 20 s, the paper points were pooled into a vial containing 2.0 ml of VMGA III transport medium. (25) The sample vials were maintained at room temperature, transferred to the laboratory and processed within 4 h after sampling. After the vials were placed in an incubator for 30 min at 37[degrees]C, bacterial plaque was mechanically dispersed with a test tube mixer at the maximal setting for 60 s. Serial 10-fold dilutions were prepared in pepton water, and aliquots were plated on MacConkey agar. The plates were incubated aerobically at 37[degrees]C for 24 h. Each isolate was characterized according to colonial and cellular morphology and Gram-stain characteristics. GNER were speciated using a standardized biochemical test (BD, Sparks, MD). TVC were defined as the total number of colony-forming units obtained on non-selective media plates. Species found on selective media were enumerated and their percentage of TVC was calculated.

Each patient provided a pooled subgingival plaque sample. Equal numbers of isolates were used from each subject.

Statistical Analysis

Data were entered into an Excel (Microsoft office 2007) database and were proofed for entry errors. The subject was the unit for the basic statistical analysis. Mean values [+ or -] SD and the proportions of sites within various categories of scoring units were calculated for data description. Normal distribution of continuous variables was verified with the Kolmogorov-Smirnov test. Categorical data were analysed with the [CHI SQUARE] test, and the percentage data between the two groups were compared with the Mann-Whitney test. Differences between groups and between different timepoints within groups were tested by the Mann-Whitney test and the Wilcoxon's rank test, respectively.

Only sites with initial PD [greater than or equal to] 4 mm were included in the statistical analyses, as shallower sites did not receive SRP, although they were treated with subgingival scaling. PD was set as the primary outcome and CAL as secondary outcome of the present study. Explanatory variables that were recorded were BOP and presence of enteric rods and periodontopathogens tested.

Data concerning sites with PD > 6 mm were analysed separately with the site as the observational unit. At each timepoint, for each group, the proportion of sites > 6 mm was calculated and changes of these proportions from baseline between groups were compared using the Mann-Whitney test.

Microbiological data were analysed with the subject as the observational unit. The number of subjects positive for the investigated species at any of the sampled sites were estimated at all timepoints. In order to identify specific differences between pairs of groups at each timepoint the Mann-Whitney test was applied, while differences between timepoints within each group were tested with the Wilcoxon's test.

The significance level was set at 0.05 for all tests.

All data handling and statistical testing were performed with a software package (SPSS, Statistical Package for the Social Sciences, version 15, Chicago, IL).

Sample size calculation

Study sample size calculations were based on subject level analyses as the study randomized individuals. The ideal sample size to assure adequate power to this clinical trial was calculated considering differences of at least 1 mm for CAL and a standard deviation of 1.1 mm between groups in initially deep periodontal pockets (> 6 mm). (6) Based on these calculations, it was determined that [greater than or equal to] 19 subjects per group would be necessary to provide an 80% power with an [alpha] of 0.05.

RESULTS

Subject retention

Ninety-eight subjects were assessed for their eligibility before entering the study. Of these, 22 subjects were excluded because they did not meet the inclusion criteria. Of the 76 subjects recruited, 72 patients had complete data for all three monitoring visits, while two subjects had one missing visit. Intent-to-treat analyses were performed in the 2 subjects with missing data, whereby the last observation was carried forward, providing a total of 74 subjects with complete data that were included in the analyses. The remaining two subjects (one from each group) had baseline data only and their data were not included in the analyses. Participation of individuals during the study is illustrated in figure 1.

[FIGURE 1 OMITTED]

Adverse events

All the subjects who completed the study reported full adherence to the prescribed course of antibiotics. Seven subjects from the CIPRO + MET group reported adverse events during the study, such as diarrhea and nausea. Moreover, three subjects from the CIPRO + MET group reported dizziness. Most subjects from the MOX group affirmed that the medications did not cause any major disturbance in their daily routine.

Clinical findings

Table 1 presents the baseline clinical and demographic characteristics of the 76 patients subdivided into the two treatment groups. There were no statistically significant differences among treatment groups for any of the parameters except for age.

Table 2 displays descriptive statistics and comparisons for both groups concerning BOP, PD and CAL. No differences were observed between the groups at any timepoint. When comparisons were made within each group, both groups resulted in significant reduction of PD and BOP compared with baseline (P < 0.001), and this difference was maintained at 6 months from baseline in both groups. No differences were observed within the groups between 3 months and 6 months after baseline. When comparisons were made within each group, both groups exhibited significant improvement in CAL compared with baseline (P < 0.001), and this difference was maintained at 6 months from baseline in both groups. In the same way, no differences were observed within the groups between 3 months and 6 months after baseline.

Additional differences concerning the effect of different treatments on the primary outcome were sought with the site instead of the subject as the observational unit, analyzing the subset of pockets with PD > 6 mm (table 3). According to the findings, subjects in both groups at 6 months displayed the greatest reduction from baseline in proportions of sites with PD > 6 mm (P < 0.001). No differences were observed between the subjects who received adjunctive MOX or CIPRO + MET.

Microbiological findings

The number of subjects positive for investigated species is presented in table 4. At baseline no differences were observed between both groups. Likewise, no differences were observed between groups at any timepoint. Administration of antimicrobials resulted in elimination and reduction of most bacteria, at the subject level, 3 months after baseline. GNER and A. actinomycetemcomitans were not identified in both groups 3 months after baseline, and P. gingivalis and T. forsythia were significantly reduced in both groups 3 months after baseline (P < 0.05). Six months from baseline, a recolonization of investigated species was observed, except for GNER and A. actinomycetemcomitans. However, P. gingivalis and T. forsythia were reduced significantly from baseline (P < 0.05).

DISCUSSION

This randomized, clinical trial evaluated the clinical and microbiological effects of SRP in combination with adjunctive antibiotics (MOX versus CIPRO+MET) in the treatment of subjects with chronic periodontitis associated with GNER. These bacteria are often recovered from the subgingival microbiota of patients considered being clinically refractory to mechanical and conventional antibiotic periodontal therapies. (12-15) The high pathogenic potential of GNER10 together with the high proportions in refractory lesions (10, 12-15) implicate them in the pathogenesis of various forms of periodontitis. (17)

This suggests the need for appropriate antibiotherapy in treating periodontal infections involving GNER. (10, 12-15) Moreover, GNER can be key pathogens in severe infections of the lower respiratory tract, skin, bone and joints, urinary tract, and various body sites (8, 11, 28) and they elaborate a number of potent enzymes and toxins of possible importance for destruction of periodontal tissues. (11)

The adjunctive use of MOX or ciprofloxacin was selected based on earlier in vitro (1, 17-21) and in vivo (4, 10) studies that suggested the clinical and microbiological benefits of these antibiotics in the treatment of periodontitis associated with GNER. To the best of our knowledge, the present study is the first clinical study comparing MOX and CIPRO + MET as an adjunct to SRP.

Clinical data

Both therapies used in the present study improved the clinical parameters evaluated. SRP combined with either MOX or with CIPRO + MET was equally effective in improving PD and CAL. The same pattern was observed for the BOP parameter and no differences were observed between groups at any time on the subject level. To our knowledge, only two studies to date have independently investigated the efficacy of adjunctive moxifloxacin (4) and ciprofloxacin alone (10) in periodontitis. With this in mind, our results are in agreement with Guentsch et al (4) who showed an enhanced clinical response when MOX was combined with SRP on the treatment of chronic periodontitis. On the other hand, it is important to note that patients included in the study with ciprofloxacin (10) had a history of refractory periodontitis associated with GNER. This finding is especially interesting because thorough mechanical treatment was unable to completely suppress these organisms in infected periodontitis lesions. (10) Similar to the current investigation, adjunctive systemic ciprofloxacin therapy demonstrated improvements in BOP, PD and CAL. The results of the current investigation are in accord with studies in the literature that suggest that systemic metronidazole alone or combined significantly improved clinical outcomes of non-surgical periodontal debridement. (1-3,6) Interestingly, Cionca et al (1) recently showed that the addition of antimicrobial agents to thorough mechanical treatment may reduce the need for further treatment.

When analyzing data at a site level, and in particular, the proportion of sites with PD >6 mm, a similar pattern was observed between groups. MOX group and CIPRO + MET group exhibited a significant reduction of the percentages of sites > 6 mm. This finding indicates a beneficial effect of these antimicrobials on deep pockets. Conclusions of the present study agree with a recently published randomized clinical trial by Guentsch et al. (4) These authors have shown that a 7-day adjunctive course of systemic MOX resulted in an additional reduction of PD in deep pockets (> 6 mm) at 6 months also in chronic periodontitis. Similar results at 12 months were shown for ciprofloxacin in patients with refractory periodontitis harboring GNER. (10) Both systematic reviews evaluating the effects of systemically administered antibiotics suggested that antibiotics provided greater benefit in subjects with more periodontal disease and at deeper periodontal sites. (2, 3)

Microbiological data

This is the first clinical study to date that has evaluated the microbiological changes that occur with the use of CIPRO+MET and the second one that has evaluated MOX in the treatment of periodontal disease, and the first one that has directly compared these two treatment protocols. We focused on determination of GNER rods and three periodontopathogens (A. actinomycetemcomitans, P. gingivalis and T. forsythia). The reasons for studying GNER were explained previously. On the other hand, it has been shown that a decrease in the counts of periodontopathogens studied in this investigation is associated with clinical improvements and these microorganisms are predictors for therapy results. (5)

Our previous paper (20) reported four species of GNER in subgingival plaque in 20 (26.31%) of 76 patients: K. pneumoniae occurred in 12 patients, Pseudomonas aeruginosa in four patients and three other species were recovered with lower prevalence. In Latin America their occurrence varies amply within studied populations, reaching a prevalence of 17.6% in Chile (29) and 67% in Santo Domingo. (30) Similar frequencies to those encountered in our study (20) were reported among Brazilians18 and Colombians. (19, 29)

Likewise, the occurrence of periodontopathogens observed in this study was similar to previous investigations. (19, 29)

It is important to note that colonies of GNER are bigger in size (19) indicating that these organisms could colonize the periodontal pockets in high proportions. On the other hand, polymerase chain reaction (PCR) detection does not take into consideration whether the sample is viable, and thus may yield a higher frequency. (19) D'Ercole et al (31) recently compared conventional culture methods and multiplex PCR for the detection of periodontopathogenic bacteria and observed that for both methods, there was a good degree of accuracy in the determination of A. actinomycetemcomitans and P. gingivalis. Similar results were obtained for T. forsythia. (32) Both culture and PCR techniques introduced many methodological problems when applied in oral microbiology, but the ideal technique for accurate detection of pathogens in subgingival plaque samples has yet to be developed. (31) As with Botero et al, (19) the present study reports on the occurrence of the microorganisms detected based on culture techniques because it allows us later to work with the cultured microorganisms. Further studies are required in order to clarify the effect of GNER on clinical parameters and response to periodontal treatment.

In agreement with the clinical results, the two groups presented favorable changes in the subgingival microbial profile after treatment. This observation was consistent with several studies, (1, 4 6) where a diminution in occurrence of the test species was significantly associated with an improvement in CAL and a decrease in PD. The most marked decrease in prevalence occurred between the baseline visit and the three months sampling. These results are in agreement with other studies that also demonstrated the adjunctive effects of these antibiotics, in reducing red complex species (1, 4-6) and GNER. (10) Interestingly, in the current study, both adjunctive antibiotic protocols reduced subgingival GNER and A. actinomycetemcomitans to undetectable levels, after three and six months. This observation is consistent with the findings described by Slots et al (10) and Guentsch et al, (4) who found out that GNER and A. actinomycetemcomitans were reduced to undetectable levels after adjunctive ciprofloxacin and MOX respectively. As it was observed in previous studies, (1, 4-6) for P. gingivalis and T. forsythia a slight rebound was detected after three months.

The treated subjects in the current study exhibited a good clinical response, suggesting that a rapid decrease in subgingival counts of periodontopathogens and GNER may be crucial for successful periodontal therapy and long-term periodontal stability. (5, 10) The longitudinal follow-up of these subjects will help to clarify this hypothesis.

The present GNER and periodontopathogens investigated were highly susceptible

to the fluoroquinolones ciprofloxacin and moxifloxacin. (20, 22) Observed concentrations of ciprofloxacin in gingival crevicular fluid of about 2.5 [micro]g/mL are many times mores elevated than levels normally obtained in plasma. (33) This may be due to improvement of quinolones in polymorphonuclear granulocytes. (33) In therapeutic doses, the drug appears in saliva at or lower than levels achieved in serum. (33) Its low activity against Gram-negative anaerobes usually limits its use in periodontics to combinations with metronidazole.10 In contrast, the novel 8-methoxy quinolone moxifloxacin has effective antimicrobial action against both Gram-negative and Grampositive anaerobes. (20, 22) Muller et al (34) showed that all tested strains of A. actinomycetemcomitans were susceptible to moxifloxacin at 0.032 [micro]g/mL. Concentrations in saliva and capillary plasma narrowly reflect equivalent concentrations in venous plasma, but primarily surpass plasma levels. (33) Quinolones are recognized for their modulation of the immune reaction, permitting the in vitro extermination of A. actinomycetemcomitans by polymorphonuclear leukocytes. (35)

Adverse events

Ten subjects (26.3%) from the CIPRO+MET group reported adverse events during the study. These results are in agreement with a systematic review (2) evaluating the effects of systemically administered antibiotics which suggested that 39% of subjects in the test group exhibited diarrhea when provided with metronidazole alone or combined. Moreover, ciprofloxacin has been associated with rare but clinically important adverse events in patients with impaired renal function (36) and various cases of "Torsades de Pointes" have been reported in the United States. (31) Conversely, subjects from the MOX group did not report adverse events during this investigation, corroborating the results of Guentsch et al. (4)

In addition to antimicrobial activity studies, the properties of moxifloxacin have been studied, showing excellent bioavailability, long half-life and good tissue penetration of this drug. (38) Furthermore, it has an excellent tolerability. (4) The pharmacokinetic properties allow a single dose treatment per day. This reduces costs and enhances the patient's compliance. (39) This is an important fact, because incomplete adherence to a 7-day adjunctive course of systemic antibiotics is associated with decreased clinical outcomes in subjects with periodontitis. (40)

The current study did have limitations. It was a randomized study, but it was not placebo controlled. Supplying placebo tablets was considered, but with the diverse dosing regimes and the dissimilar variety of tablets/capsules implicated, composing a standard placebo offered some complexity. It was therefore decided not to use placebos. However, the examining clinician was unaware of the treatment group to which subjects were assigned. Similar situations were presented by Haffajee et al (6) and Guentsch et al. (4) in two recent clinical trials. Another group including only mechanical treatment was not included in the present study. The authors believe it is clear that the administration of systemic antibiotics during mechanical periodontal treatment is widely reported and accepted by distinguished researches during the treatment of patients with chronic periodontitis harboring GNER. (10,12-15) Moreover, two systematic reviews showed that adjunctive use of systemically administered antibiotics provided a better clinical outcome, particularly in terms of PD reduction and attachment-level gain, than SRP, not only in aggressive forms of periodontitis but also in subjects with chronic periodontitis. (2,3) Finally, it was recently reported that the addition of antimicrobial agents to thorough mechanical treatment may reduce the need for further treatment. (1) Therefore, a mechanical-treatment-only group was not used in the present protocol. Despite its limitations, a prospective decision to incorporate adjunctive MOX or CIPRO-MET as a treatment option is confirmed by the outcomes of the present study.

The current protocols for antibiotic treatment in periodontal therapy are based on microbiological studies conducted outside Latin American countries.

Consequently, the prevalence of considerable differences in the composition of the subgingival flora in diverse geographical populations may increase the possibility of developing antibiotic resistance or promoting a subgingival overgrowth of superinfecting organisms. (41) Moxifloxacin offers several advantages over the previously available fluoroquinolones including reduced propensity to promote the development of resistance. (11) Although the published data and clinical experience with these agents is limited, given their relatively recent entry into our market, this perspective attempts to provide an understanding of the potential role of moxifloxacin in the treatment of patients with chronic periodontitis harboring enteric rods in subgingival plaque.

CONCLUSIONS

Within the limitations of this study, the results indicate that patients with chronic periodontitis harboring subgingival Gram-negative enteric rods may benefit from systemic administration of moxifloxacin or ciprofloxacin plus metronidazole as an adjunctive antimicrobial therapy to periodontal mechanical treatment. Both treatment strategies resulted in significant gain of clinical attachment level and decrease in periodontal probing depth. The same pattern was observed for BOP. Suppression of GNER below detectable levels for at least 6 months was also demonstrated. Consequently, systemic use of moxifloxacin or ciprofloxacin plus metronidazole as an adjunct to conventional therapy may be an alternative for the treatment of patients with chronic periodontitis. The treatment method should be based on compliance and potential adverse effects.

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(30.) Slots J, Rams TE, Feik D, Taveras HD, Gillespie GM. Subgingival microflora of advanced periodontitis in the Dominican Republic. J Periodontol 1991; 62: 543-547.

(31.) D'Ercole S, Catamo G, Tripodi D, Piccolomini R. Comparison of culture methods and multiplex PCR for the detection of periodontophatogenic bacteria in biofilm associated with severe forms of periodontitis. New Microbiol 2008; 31: 383-391.

(32.) Boutaga K, van Winkelhoff AJ, Vandenbroucke-Grauls CM, Savelkoul PH. Periodontal pathogens: a quantitative comparison of anaerobic culture and real-time PCR. FEMS Immunol Med Microbiol 2005; 45: 191-199.

(33.) Conway TB, Beck FM, Walters JD. Gingival fluid ciprofloxacin levels at healthy and inflamed human periodontal sites. J Periodontol 2000; 71: 1448-1152.

(34.) Muller HP, Holderrieth S, Burkhardt U, Hoffler U. In vitro antimicrobial susceptibility of oral strains of Actinobacillus actinomycetemcomitans to seven antibiotics. J Clin Perio dontol 2002; 29: 736-742.

(35.) DalhoffA. Immunomodulatory activities of fluoroquinolones. Infection 2005; 33(Suppl. 2): 55-70.

(36.) Iannini PB. The safety profile of moxifloxacin and other fluoroquinolones in special patient populations. Curr Med Res Opin 2007; 23: 1403-1413.

(37.) Frothingham R. Rates of torsades de pointes associated with ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, and moxifloxacin. Pharmacotherapy 2001; 21: 1468-1472.

(38.) Cachovan G, Nergiz I, Thuss U et al. Penetration of moxifloxacin into rat mandibular bone and soft tissue. Acta Odontol Scand 2009; 67: 182-186.

(39.) Krasemann C, Meyer J, Tillotson G. Evaluation of the clinical microbiology profile of moxifloxacin. Clin Infect Dis 2001; 32 Supl 1: S51-63.

(40.) Guerrero A, Echeverria JJ, Tonetti MS. Incomplete adherence to an adjunctive systemic antibiotic regimen decreases clinical outcomes in generalized aggressive periodontitis patients: a pilot retrospective study. J Clin Periodontol 2007; 34: 897-902.

(41.) Pinheiro ET, Gomes BP, Drucker DB, Zaia AA, Ferraz CC, Souza-Filho FJ. Antimicrobial susceptibility of Enterococcus faecalis isolated from canals of root filled teeth with periapical lesions. Int Endod J 2004; 37: 756-763.

ISABEL CRISTINA GUZMAN ZULUAGA [2], HUGO GRISALES ROMERO [3], CARLOS MARTIN ARDILA MEDINA [4]

[1] Parte de este estudio fue patrocinado por una beca de la Escuela Nacional de Salud Publica y el Grupo de Epidemiologia de la Universidad de Antioquia. Los autores informan que no existe conflicto de interes relacionado con este estudio.

[2] Periodoncista, profesora asistente, Facultad de Odontologia, Universidad de Antioquia, Colombia.

[3] Ph. D. en Epidemiologia, profesor titular, Facultad de Salud Publica, Universidad de Antioquia, Colombia.

[4] Periodoncista, Ph. D., profesor asociado, Facultad de Odontologia, Universidad de Antioquia, Colombia. Director Grupo de Estomatologia Biomedica, Universidad de Antioquia, Medellin, Colombia.

RECIBIDO: MARZO 11/2011-ACEPTADO: SEPTIEMBRE 13/2011

[1] This study was sponsored in part by a grant from the National Public Health School and the Epidemiology Group of the Universidad de Antioquia. The authors report no conflict of interest related to this study.

[2] Periodontist, Assistant Professor, School of Dentistry, Universidad de Antioquia, Colombia.

[3] Ph.D. in Epidemiology, Titular Professor National Public Health School Universidad de Antioquia, Colombia.

[4] Periodontist, Ph. D. Associate Professor School of Dentistry Universidad de Antioquia, Colombia. Director, Group of Biomedical Stomatology, Universidad de Antioquia, Medellin, Colombia.

SUBMITTED: MARCH 11, 2001-ACCEPTED: SEPTEMBER 13, 2011

CORRESPONDENCIA

Carlos M. Ardila

Calle 64 No. 52-59 Medellin, Colombia.

Tel: (57-4) 219 67 60

Correo electronico: martinardila@gmail.com

CORRESPONDING AUTOR

Carlos M. Ardila

Calle 64 No. 52-59 Medellin, Colombia.

Fax 057-4-2196760

e-mail: martinardila@gmail.com
Tabla 1. Caracteristicas clinicas y demograficas de los sujetos con
los datos de los dos grupos de tratamiento al inicio del estudio

Parametro                   grupo MOX N = 38   grupo CIPRO + MET N = 38

Edad (promedio [+ o -] DE)   45 [+ o -] 7,3         47 [+ o -] 8,6
Sexo, mujeres                   22 (58%)               23 (60%)
Fumadores                        8 (21%)               9 (24%)
Placa (% [+ o -] DE)          58 [+ o -] 30         59 [+ o -] 30

Parametro                        p-valor

Edad (promedio [+ o -] DE)  0,01 Mann Whitney
Sexo, mujeres               0,1 Chi cuadrado
Fumadores                   0,7 Chi cuadrado
Placa (% [+ o -] DE)        0,5 Mann Whitney

DE = desviacion estandar.

Table 1. Clinical and demographic features of subjects with data for
the two treatment groups at baseline

Parameter                   MOX group N = 38   CIPRO + MET group N = 38

Age mean [+ or -] SD         45 [+ or -] 7.3       47 [+ or -] 8.6
Gender, Females                 22 (58%)               23 (60%)
Smokers, current                 8 (21%)               9 (24%)
Plaque (% [+ or -] SD)       58 [+ or -] 30         59 [+ or -] 30

Parameter                        p-value

Age mean [+ or -] SD        0.01 Mann Whitney
Gender, Females             0.1 [CHI SQUARE]
Smokers, current            0.7 [CHI SQUARE]
Plaque (% [+ or -] SD)      0.5 Mann Whitney

SD = standard deviation.

Tabla 2. Parametros clinicos de los dos grupos durante el periodo
experimental

Parametro       GRUPO MOX             GRUPO
              Linea de base          3 meses             6 meses

SS            72 [+ o -] 28       15 [+ o -] 4        13 [+ o -] 4
PS          5,15 [+ o -] 0,6    3,13 [+ o -] 0,4    3,12 [+ o -] 0,3
NIC         5,25 [+ o -] 0,6    3,84 [+ o -] 0,7    3,71 [+ o -] 0,7

Parametro   GRUPO CIPRO + MET         GRUPO
              Linea de base          3 meses             6 meses

SS            74 [+ o -] 27       16 [+ o -] 5        14 [+ o -] 4
PS          5,14 [+ o -] 0,5    3,12 [+ o -] 0,3    3,12 [+ o -] 0,3
NIC         5,23 [+ o -] 0,6    3,82 [+ o -] 0,7     3,7 [+ o -] 0,7

No se observaron diferencias entre los grupos (Mann-Whitney p > 0,05).

Se observaron diferencias al interior de los grupos entre la linea de
base y los 3 meses, y entre la linea de base y los 6 meses (Wilcoxon
p < 0,05)

No se observaron diferencias entre los grupos (Wilcoxon p < 0,05)

Table 2. Clinical parameters of the two groups during the
experimental period

Parameter          MOX                Group

                Baseline            3 months            6 months

BOP          72 [+ or -] 28       15 [+ or -] 4       13 [+ or -] 4
PD          5.15 [+ or -] 0.6   3.13 [+ or -] 0.4   3.12 [+ or -] 0.3
CAL         5.25 [+ or -] 0.6   3.84 [+ or -] 0.7   3.71 [+ or -] 0.7

Parameter       CIPRO+MET             Group

                Baseline            3 months            6 months

BOP           74 [+ or -] 27      16 [+ or -] 5       14 [+ or -] 4
PD          5.14 [+ or -] 0.5   3.12 [+ or -] 0.3   3.12 [+ or -] 0.3
CAL         5.23 [+ or -] 0.6   3.82 [+ or -] 0.7   3.70 [+ or -] 0.7

SD = standard deviation.

No differences were observed between groups at any time (Mann-
Whitney test p > 0.05).

Differences were observed within groups between baseline and 3
months, and between baseline and 6 months (Wilcoxon's test p < 0.05).

No differences were observed within the groups between 3 months and 6
months after baseline (Wilcoxon's test p < 0.05).

Tabla 3. Cambio de proporciones de los sitios con profundidad de
sondaje > 6 mm en los dos grupos durante el periodo experimental

                                                            CIPRO +
                                                             METRO
                              MOX Cambio                    Cambio
                  Grupo N     desde linea     Grupo N     desde linea
Observacion      (sitios)      base (%)      (sitios)      base (%)

Linea de base       261           --            264           --
3 meses             24           90,1           27           89,7
6 meses             21           91,9           24           90,9

No se observaron diferencias entre los grupos (Mann-Whitney p > 0,05).
Se observaron diferencias entre los grupos (Mann-Whitney p > 0,05).

Table 3. Change of proportions of sites with probing depth > 6 mm of
the two groups during the experimental period

                                                             Group
                                 Group                       Group
                              Change from                 Change from
                   MOX n       Baseline     CIPRO+METRO    Baseline
Observation       (sites)         (%)        n (sites)        (%)

Baseline            261           --            --            --
3 months            24           90.1           27           89.7
6 months            21           91.9           24           90.9

No differences were observed between two groups at any time (Mann-
Whitney p > 0.05).

Differences were observed within groups between baseline and 3
months, and between baseline and 6 months (Wilcoxon's test p < 0.05).

Tabla 4. Numero de sujetos positivos para bacilos entericos Gram
negativos (Enteric), Aggregatibacter actinomycetemcomitans (A. a.),
Porphyromonas gingivalis (P g.) y Tanerella forsythia (T. f.) durante
el periodo experimental

                  Enteric                     a

Grupos              MOX       CIPRO + MET    MOX    CIPRO + MET

Linea de base       11             9          9          9
3 meses              0             0          0          0
6 meses              0             0          0          0

                                 T. f

Grupos             P. g       CIPRO + MET    MOX    CIPRO + MET

Linea de base       MOX           26         21         22
3 meses                           10          8          9
6 meses             25            13         10         11
                     9
No se observaron    11

No se observaron diferencias entre los grupos (Mann-Whitney p > 0,05).

P g and T.f se redujeron significativamente en los dos grupos despues
de 3 y 6 meses (Wilcoxon p < 0,05).

Table 4. Number of subjects positive for Gram negative enteric rods
(Enteric), Aggregatibacter actinomycetemcomitans (A. a),
Porphyromonas gingivalis (P. g.) and Tanerella forsythia (T. f.)
during the experimental period

                  Enteric                   A. a

Groups              MOX        CIPRO+MET     MOX     CIPRO+MET

Baseline            11             9          9          9
3 months             0             0          0          0
6 months             0             0          0          0

                   P. g                     T. f.

Groups              MOX        CIPRO+MET     MOX     CIPRO+MET

Baseline            25            26         21         22
3 months             9            10          8          9
6 months            11            13         10         11

No differences were observed between both groups at baseline
(Mann-Whitney test p > 0.05).

No differences were observed between groups at any time point
(Mann-Whitney test p > 0.05).

P. g. and T.f. were significantly reduced in both groups 3 months and
6 months after baseline (Wilcoxon's signed ranks test p < 0.05).
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Publication:Revista Facultad de Odontologia
Date:Aug 1, 2011
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