Comparación de métodos histoquímicos para el análisis histológico de muestras de encía humana / Comparison of histochemical methods for the histological analysis of human gingival samples

Introducción: Las técnicas de coloración histológica son útiles en el análisis ultraestructural de muestras tisulares, incluyendo el tejido gingival. Objetivo: Comparar la utilidad de tres métodos histoquímicos (hematoxilina-eosina, Masson Goldner y rojo sirio) en la identificación de elementos celulares y otros constituyentes del tejido gingival. Métodos: Estudio experimental in vitro que comprendió el análisis de tejidos gingivales de donantes sanos sin signos de inflamación gingival y con indicación de cirugía periodontal. Las muestras de encía se obtuvieron mediante gingivectomía, se procesaron e incluyeron en parafina, posteriormente se realizaron cortes con un micrótomo y se depositaron en portaobjetos de adhesión con polisina. Las muestras se agruparon y fueron teñidas con hematoxilina-eosina, Masson Goldner y rojo sirio, finalmente fueron visualizadas en un microscopio óptico Leica DM 750®. La lectura de los hallazgos fue realizada por patólogos orales. Resultados: La coloración hematoxilina-eosina evidencia elementos celulares y extracelulares del tejido epitelial y conectivo. Núcleos de color azul violeta, citoplasmas rosados, fibras de colágeno de matiz rosa claro, arteriolas y vénulas con túnica adventicia, media e íntima diferenciadas. La coloración Masson Goldner diferencia núcleos de coloración púrpura y citoplasma fucsia, presenta especificidad en identificar fibras de colágeno con tonalidad verde, distribuidas densa, homogénea y paralelamente en el tejido conectivo gingival. La tinción rojo sirio, permitió identificar las fibras de colágeno de color rosa brillante, mientras que el tejido epitelial y los vasos sanguíneos fueron de color amarillo. Conclusión: Cada coloración histológica evaluada en el presente trabajo tiene cierta afinidad y sensibilidad por estructuras celulares y componentes de la matriz extracelular específica. Su empleo es útil en el estudio de tejidos gingivales y podrían contribuir en el análisis de biopsias gingivales(AU)

Introduction: Histological staining techniques are useful in the ultrastructural analysis of tissue samples, including gingival tissue. Objective: Compare the usefulness of three histochemical methods (hematoxylin-eosin, Masson-Goldner and sirius red) for identification of cellular elements and other constituents of gingival tissue. Methods: An in vitro experimental study was conducted which included the analysis of gingival tissue from healthy donors without gingival inflammation signs and indication of periodontal surgery. The gum samples were obtained by gingivectomy, processed with paraffin, cut with a microtome and placed on Polysine adhesion slides. The samples were grouped, stained with hematoxylin-eosin, Masson Goldner and sirius red, and visualized under a Leica DM 750® microscope. Reading of the findings was performed by oral pathologists. Results: Hematoxylin-eosin staining found cellular and extracellular elements of the epithelial and connective tissue: violet-blue nuclei, pink cytoplasms, light rose collagen fibers, and arterioles and venules with differentiated tunica adventitia, media and intima. Masson-Goldner staining differentiated purple nuclei and fuchsia cytoplasm. It displayed specificity identifying green collagen fibers with dense, homogeneous and parallel distribution in the gingival connective tissue. Sirius red staining allowed identification of bright rose collagen fibers, whereas epithelial tissue and blood vessels were yellow. Conclusion: Each of the histological staining methods evaluated in the study shows a certain affinity with and sensitivity to cellular structures and components of the specific extracellular matrix. All three are useful for the study of gingival tissue and could contribute to the analysis of gingival biopsies(AU)

Validation of a new experimental model of extrusive luxation on maxillary molars of rats: a histological study.

BACKGROUND/AIM: The aim of this study was to test and validate a new model of extrusive luxation trauma on maxillary first molars of rats. MATERIAL AND METHODS: Forty adult male rats (Rattus norvegicus albinus, Wistar; weight = 230-250 g), 45 days old, were divided into eight groups (n = 5): control groups, in which animals were not subjected to any procedure and waited 1 day (GC1D) or 3 days (GC3D) for euthanasia, and experimental groups, in which animals were subjected to forces of 1100cN, 1300cN, or 1500cN and waited 1 or 3 days for euthanasia (GT1100/1D, GT1100/3D, GT1300/1D, GT1300/3D, GT1500/1D, GT1500/3D). In animals of the experimental groups, trauma was produced by an extrusive force in maxillary first right molars. Four-micrometer serial cuts stained with hematoxylin and eosin (HE) were made. Descriptive microscopic analysis of first upper right molar and semi-quantitative analysis (scores 1 to 4) of intensity of acute and chronic inflammation and vascular changes in the periodontal ligament and active and inactive external root resorption were conducted. The distribution of scores in the groups was compared using the Freeman-Halton extension of Fisher's exact test. The significance level was 5%. RESULTS: It was observed that vascular disorders (bleeding) on the periodontal ligament became more evident with increasing extrusive force. CONCLUSIONS: This new method was capable of generating histological changes, proving its secure application in this research area. The 1500cN force produced more damage on the periodontal ligament. CLINICAL RELEVANCE: The validation of a new experimental method can produce more reliable evidence in further research.

Effect of low-intensity therapeutic ultrasound on wound healing in rats subjected to third-degree burns.

PURPOSE: To determine the effectiveness of low-intensity therapeutic ultrasound (LITUS) on wound healing in rats with third-degree burns. METHODS: Twenty rats were divided into the Control Group that comprised four rats without third-degree burns that did not undergo LITUS, the Burned Group (BG), comprising eight rats with third-degree burns that did not undergo LITUS, and the Burned with Treatment Group (BTG), comprising eight rats with third-degree burns that were administered LITUS. LITUS began 24 h after injury and involved daily applications for 8 min at 0.1 W/cm2 for 14 days. RESULTS: The BTG lost less weight than the BG (Q=2.75; p<0.05). No visible differences were apparent among the groups' lesions on day 4. By the end of treatment, wound healing was more evident in the BTG. No statistically significant differences were found between the BG and the BTG in relation to the parameters measured using the histological changes in burn wound healing scoring system. CONCLUSION: The LITUS protocol applied to the animals with third-degree burns accelerated the formation of fibrin-leukocyte crusts and significantly reduced weight loss. However, burn wound healing was not accelerated.

Effect of low-intensity therapeutic ultrasound on wound healing in rats subjected to third-degree burns

PURPOSE: To determine the effectiveness of low-intensity therapeutic ultrasound (LITUS) on wound healing in rats with third-degree burns. METHODS: Twenty rats were divided into the Control Group that comprised four rats without third-degree burns that did not undergo LITUS, the Burned Group (BG), comprising eight rats with third-degree burns that did not undergo LITUS, and the Burned with Treatment Group (BTG), comprising eight rats with third-degree burns that were administered LITUS. LITUS began 24 h after injury and involved daily applications for 8 min at 0.1 W/cm2 for 14 days. RESULTS: The BTG lost less weight than the BG (Q=2.75; p<0.05). No visible differences were apparent among the groups' lesions on day 4. By the end of treatment, wound healing was more evident in the BTG. No statistically significant differences were found between the BG and the BTG in relation to the parameters measured using the histological changes in burn wound healing scoring system. CONCLUSION: The LITUS protocol applied to the animals with third-degree burns accelerated the formation of fibrin-leukocyte crusts and significantly reduced weight loss. However, burn wound healing was not accelerated.

Acupuncture increases the diameter and reorganisation of collagen fibrils during rat tendon healing.

BACKGROUND: Our previous study showed that electroacupuncture (EA) increases the concentration and reorganisation of collagen in a rat model of tendon healing. However, the ultrastructure of collagen fibrils after acupuncture is unknown. OBJECTIVES: To assess the effect of acupuncture protocols on the ultrastructure of collagen fibrils during tendon healing. METHODS: Sixty-four rats were divided into the following groups: non-tenotomised (normal group), tenotomised (teno group), tenotomised and subjected to manual acupuncture at ST36 (ST36 group), BL57 (BL57 group) and ST36+BL57 (SB group) and EA at ST36+BL57 (EA group). The mass-average diameter (MAD) and the reorganisation of collagen fibril diameters were determined during the three phases of tendon healing (at 7, 14 and 21 days). RESULTS: The MAD increased during the three phases of healing in the SB group. In the EA group, MAD increased initially but was reduced at day 21. The reorganisation of collagen fibrils was improved in the EA and SB groups at days 14 and 21, respectively. EA at day 21 appeared to reduce the reorganisation. CONCLUSIONS: These results indicate that the use of EA up to day 14 and manual acupuncture at ST36+BL57 up to day 21 improve the ultrastructure of collagen fibrils, indicating strengthening of the tendon structure. These data suggest a potential role for acupuncture in rehabilitation protocols.

The effect of muscle actions on the level of connective tissue damage.

The aim of this study was to compare the effect of concentric with eccentric muscle actions on the resulting level of damage to connective tissues by urinary concentration of hydroxyproline. Twenty-one male volunteers were divided into control group (CG), experimental concentric group (ECG), and experimental eccentric group (EEG). The measures of hydroxyproline were performed at three times: pretest, fourth week, and posttest. Biceps curl and chest press exercises also were performed with three sets of 10 repetitions two times per week for both experimental groups. Analysis of variance (ANOVA) showed a significant difference between pretest of the CG and pretest of the ECG (p = 0.002), and between pretest and posttest for the EEG (p = 0.029). Therefore, this study concluded that the level of damage to the connective tissue is greater when exercises involving eccentric muscle actions are performed. The continuity of training, however might reduce this damage.

Soft tissue pathology around the hip.

Snapping hip syndromes have been treated with open surgery for many years. Recently, endoscopic techniques have been developed for treatment of snapping hip syndromes with results that are at least comparable if not better than those reported for open procedures. The greater trochanteric pain syndrome is well known by orthopedic surgeons. However, deep understanding of the pathologic conditions generating pain in the greater trochanteric region and endoscopic access to it has only recently been described. Although evidence regarding endoscopic techniques for the treatment of the greater trochanteric pain syndrome is mainly anecdotal, early published reports are encouraging.

Morphological changes in distant muscle fibers following thermal injury in Wistar rats / Alterações morfológicas das fibras musculares de um músculo distante da lesão térmica em ratos Wistar

PURPOSE: Thermal injury causes catabolic processes as the body attempts to repair the damaged area. This study evaluated the effects of a scald injury on the morphology of muscle fibers belonging to a muscle distant from the lesion. METHODS: Thirty Wistar rats were divided into control (C) and scalded (S) groups. Group S was scalded over 45 percent of the body surface, standardized by body weight. Rats in both groups were euthanized at four, seven and 14 days following the injury. The middle portions of the medial gastrocnemius muscles were sectioned, stained with hematoxylin and eosin and Picrosirius, and submitted to histological analysis. RESULTS: Control group sections exhibited equidistantly distributed polygonal muscle fibers with peripheral nuclei, characteristic of normal muscle. The injured group sections did not consistently show these characteristics; many fibers in these sections exhibited a rounded contour, variable stain intensities, and greater interfiber distances. A substantially increased amount of connective tissue was also observed on the injured group sections. CONCLUSION: This experimental model found a morphological change in muscle distant from the site of thermal injury covering 45 percent of the body surface.(AU)

OBJETIVO: A lesão térmica conduz o organismo a processos catabólicos a fim de reparar a área lesada. O propósito do presente estudo foi avaliar os efeitos da lesão térmica por escaldadura na morfologia das fibras musculares de um músculo distante da lesão. MÉTODOS: Foram utilizados 30 ratos Wistar, divididos em: Grupo submetido à lesão térmica por escaldadura (S) e Grupo Controle (C). A escaldadura foi provocada em 45 por cento da superfície corporal dos animais e padronizada pelo peso corpóreo. A eutanásia ocorreu nos períodos pós-lesão de quatro, sete e 14 dias. Retirou-se a parte média dos músculos gastrocnêmio medial os quais foram submetidos à técnica histológica de Hematoxilina-Eosina e Picrosírius. RESULTADOS: O aspecto poligonal das fibras musculares, com núcleos periféricos, bem como a distribuição eqüidistante das mesmas, característico das descrições da musculatura normal, verificado nos animais do grupo C não foi uma constante nas fibras do músculo dos animais do grupo S. Nestes, muitas fibras exibiram contorno arredondado, diferentes intensidades de marcação bem como maior distanciamento entre as mesmas. Observou-se ainda aumento substancial da quantidade de tecido conjuntivo no grupo S. CONCLUSÃO: Houve alteração da morfologia do músculo distante da lesão como resposta à lesão térmica de 45 por cento em modelo experimental.(AU)

Avaliação do efeito do cloridrato de papaverina na reparação de feridas cirúrgicas abertas em dorso de ratos / Effect of papaverine hydrochloride in open surgical repair of wounds in rats

Os objetivos deste estudo foram avaliar o efeito da aplicação subcutânea do cloridrato de papaverina no processo de reparação de feridas cirúrgicas abertas no dorso de ratos e avaliar pelos métodos histomorfológico, histomorfométrico e imunoistoquímico eventos biológicos do processo de reparação. Foram realizadas feridas dérmicas padronizadas com punch, 5mm de diâmetro e 2mm de profundidade, no dorso de ratos. Os animais foram divididos em dois grupos conforme o tratamento realizado: no grupo controle, 25 ratos foram tratados através da injeção de cloreto de sódio 0,9% e no grupo teste, 25 ratos foram tratados através da injeção da solução de cloridrato de papaverina a 50mg/mL de cloreto de sódio a 0,9%. Em ambos os grupos foi aplicado subcutâneo (por quadrante da ferida), 0,25mL da solução correspondente aos frascos do grupo teste ou grupo controle totalizando 1 mL da solução. Os 50 espécimes foram processados para as análises macro e microscópica. Para análise do cálculo do edema, foram utilizados 10 ratos (5 animais do grupo teste e 5 animais do grupo controle). Fragmentos de pele padronizados (3cm2) foram removidos da área da ferida e pesados no período de 6 horas. Para análise morfométrica do fechamento da ferida/formação de cicatriz, 10 ratos foram utilizados (5 animais do grupo teste e 5 do grupo controle). As feridas cirúrgicas padronizadas foram fotografadas nos períodos de 0h, 3,7,14 e 21 dias pós-cirúrgico e as imagens foram analisadas por software de morfometria (ImageLab2000®) quanto a área, perímetro e fator de forma. Pela técnica de coloração da hematoxilina e eosina, a análise histomorfológica (análise qualitativa descritiva) e histomorfométrica (análise quantitativa em relação à reepitelização, formação do tecido de granulação, edema, celularidade e matriz colagênica) foram realizadas nos períodos de 6h, 3,7,14 e 21 dias e analisadas sob microscopia de luz

Reações de imunoistoquímica com o anticorpo anti-actina de músculo liso foi realizada para identificação e contagem do número de miofibroblastos nos períodos de 3, 7, 14 e 21 dias. A partir do modelo experimental avaliado, comprovou-se a ação vasodilatadora da papaverina. Os fragmentos padronizados do grupo teste apresentaram maior peso em relação ao grupo controle (p= 0,047). As feridas do grupo teste mostraram um fechamento maior e menor formação de cicatriz quando comparado ao grupo controle no período de 21 dias. No grupo teste, uma maior quantidade de edema (p = 0,028) e uma menor quantidade de matriz colagênica (colágeno) (p = 0,028) foram encontradas no período de 6 horas. Houve maior reepitelização no grupo teste no período de 7 dias e menor formação de tecido de granulação nos períodos de 14 e 21 dias para o mesmo grupo...

The objectives of this study were to evaluate the effect of subcutaneous administration of papaverine hydrochloride in the process of open surgical repair of wounds in the back of rats and evaluate the methods histomorphological, immunohistochemical and histomorphometric biological events of the repair process. Standardized wounds were inflicted by dermal punch, 5mm in diameter and 2mm deep, in the backs of rats. Animals were divided into two groups according to treatment: control group, 25 rats were treated by injection of sodium chloride 0.9%) and test group (25 rats treated by the injection of papaverine hydrochloride 50 mg / mL sodium chloride 0.9%). In both groups was administered subcutaneously (by quadrant of the wound), 0.25 mL of the solution corresponding to bottles of the test group or control group, totaling 1 mL of the solution. The 50 specimens were processed for macro and microscopic analysis. For analysis of the calculation of edema, we used 10 rats (5 animals in the test group and 5 control animals). Standardized skin fragments (3cm2) were removed from the wound area and heavy during the 6 hours. For morphometric analysis of the closure of the wound / scar formation, 10 rats were used (five animals in the test group and 5 in the control group). Surgical wounds were photographed in standardized periods of 0h, 3,7,14 and 21 days after surgery and the images were analyzed by morphometry software (ImageLab2000 ®) as area, perimeter and form factor

By staining with hematoxylin and eosin, the histomorphologic analysis (descriptive qualitative analysis) and histomorphometric (quantitative analysis in relation to reepithelialization, formation of granulation tissue, edema, cellularity and collagen matrix) were recorded at 6h, 3.7 , 14 and 21 days and analyzed under light microscopy. Immunohistochemical reactions with anti-smooth muscle actin was performed to identify and count the number of myofibroblasts at 3, 7, 14 and 21 days. From the experimental model evaluated, proved the vasodilator papaverine. The fragments of the standardized test group had a higher weight in the control group (p = 0.047). The wounds of the test group showed a greater closing and less scarring compared to control group within 21 days. In the test group, a greater amount of edema (p = 0.028) and a smaller amount of collagen matrix (collagen) (p = 0.028) were found within 6 h. A greater reepithelialization in the test group after 7 days and less formation of granulation tissue during periods of 14 and 21 days for the same group. In the control group, a greater number of myofibroblasts was found when compared to the test group during the periods of 14 and 21 days (p = 0, 016). Within the limits of this study, subcutaneous administration of papaverine hydrochloride appears to accelerate the repair of surgical wounds opened in the back of rats. Its application promotes vasodilation and a greater inflammatory exudate in the early repair process...

Recubrimiento de las recesiones gingivales con la técnica de injerto conjuntivo subepitelial asociado al deslizamiento coronario del colgajo: relato de caso clínico / Coronal positioned flaps associated with free subepitelial conective tissue graft for treatment of gingival recession: clinical case report

Recesión gingival es definida como la localización apical del margen gingival en relación a la unión cemento - esmalte, siendo predominante en la cara vestibular de los dientes. Además de provocar una desarmonía en la estética, puede causar sensibilidad destinaría. Para el tratamiento de recesiones gingivales han sido propuestas técnicas que pueden ser agrupadas en injertos pediculados, injertos libres y regeneración de tejidos guiada. El objetivo de este trabajo es relatar el caso clínico de un paciente con recesiones gingivales generalizadas. Luego de la anamnesis y examen clínico, se atribuye la etiología de las recesiones al cepillado incorrecta y trauma oclusal. Se realizó una adecuada instrucción de higiene bucal y ajuste oclusal. Se procedió a la aplicación de nitrato de potasio al 5 por ciento por dos meses, sin embargo, él paciente se quejaba de sensibilidad y del aspecto estético. Para el recubrimiento de las raíces expuestas fueron realizadas las técnicas de colgajo posicionado coronalmente asociado y no asociado al injerto conjuntivo subepitelial. Luego de un año los dientes muestran ausencia de sangramiento y sensibilidad destinaría, con profundidad de sondaje normal (<3mm). Los resultados de esta técnica se muestran satisfactorios, atendiendo las expectativas del paciente

Gingival recession is defined as the apical location of the gingival in relation to the cement-enamel junction, being predominant in buccal aspect. Other than causing esthetic disharmony, it can cause dentine sensitive. To treat gingival recession, the following techniques can be employed: pedicle gingival graft, free gingival graft, and guided tissue regeneration. The purpose of this study is to present a case report with generalized gingival recession. After anamneses and clinical exam, the etiology of the gingival recession was indicated as incorrect oral hygiene and occlusal trauma. The patient receive adequate oral hygiene instruction and the occlusion was adjusted. Five percent potassium nitrate was applied locally for two months, however, the patient still complained of dentine sensitivity and lack of esthetics. For root coverage, coronal positioned flaps were conduct with or without free subepithelial connective tissue. One year later the teeth presented absence of bleeding and dentine sensitive, with normal probe depth (<3mm). The results of this techniques fulfilled the patients esthetic need and eliminated the dentinal sensitivity

Histomorphometric study of peri-implant bone healing in the case of nerve injury: an experimental model in rats.

Causes of dental implant failure are of more than passing interest. Within the group of failures caused by iatrogenic factors, injury to the epineurium has been reported to cause the formation of peri-implant fibrous tissue (fibrointegration). The aim of this study was to perform a histomorphometric evaluation of the percentage of osseointegration of implants in contact with the epineurium. Twenty Wistar rats were used. The first lower molars were extracted under xylazine-ketamine anesthesia. A titanium screw implant (diameter, 0.75 mm; length, 2.26 mm) was placed. In the control group (n = 10), apical anchorage of the implant was performed. In the experimental group (n = 10), the apical portion of the implant was placed in contact with the epineurium of the lower mandibular nerve. All animals were killed by ether overdose 30 days after implantation. Radiographs were taken, and the samples were processed for embedding in acrylic resin. Ground sections were obtained along the vestibulo-lingual axis of the mesial alveolus that contained the implant and were stained with toluidine blue. The histologic analysis revealed the presence of bone tissue in the apical portion of the control group samples. In the experimental group, the implant was in contact with the epineurium. There were no statistically significant differences in the percentage of osseointegration between both groups (control group, 39% +/- 9%; experimental group, 38% +/- 10%). The results obtained with this experimental model show that the contact of the implant with the epineurium would not impair the process of osseointegration.

The effects of local trauma to the enamel-related periodontal tissues in the eruption of the rat incisor.

The periodontal tissues related to enamel (PTE) of the rat incisor comprise a connective tissue derived from the dental follicle and the enamel organ with its successive stages of development. Localized damage to these tissues in rat lower incisors was done surgically in three ways: with an endodontic file introduced into the labial periodontal space through either (i) its basal or (ii) its incisal extremities, or (iii) by the partial removal of the mandibular lower border, at the level of the molar teeth, together with the introduction of an endodontic file into the incisal part of that space. The lesions in the molar region of the PTE produced first a variable period of retarded eruption, and, depending upon their extent or degree were followed by a cessation of the eruptive movement and, in the majority of the operated teeth, a recovery of the normal eruption rate before the end of the experiment (17 weeks after surgery). Access to the PTE through the basal portion of the socket was erratic, but when the tissues were damaged produced similar effects. Effects on eruption of lesions produced through the alveolar crest were minimal or even absent. Localized injury to the periodontal ligament of either lower or upper incisors did not produce similar effects on tooth eruption. The dental follicle and the enamel organ of teeth of limited growth when their crown is completed are similar to the PTE in the molar region of continuously growing rodent incisors. In teeth of limited growth these tissues play an essential part in the intraosseous stage of eruption. The results here suggest that the PTE may also have a role in the supraosseous stage of eruption, which is continuous in teeth such as rat incisors due to the presence of a continuously functioning odontogenic organ.

Cambios histopatológicos en el tejido gingival enfermo / Histopathological changes in the diseased gingival tissue

Se realizó un estudio descriptivo y transversal en pacientes que acudieron al Servicio de Periodoncia de la Clínica Estomatológica Provincial Docente de Santiago de Cuba, cuyo universo estuvo constituído por 22 pacientes necesitados de tratamiento quirúrgico. El objetivo de este trabajo fue el de determinar los cambios epiteliales y del corium más frecuentes y relacionarlos con los aspectos clínicos y el diagnóstico de la enfermedad periodontal, así como precisar la frecuencia de invasión bacteriana en el tejido gingival enfermo. En un formulario confeccionado al efecto, se anotaron: aspectos clínicos de la encía, diagnóstico clínico y cambios histopatológicos. Entre los principales hallazgos se encuentran que el cambio epitelial más frecuente resultó ser la acantosis, y que la invasión bacteriana se puso de manifiesto en el 13,6 por ciento de los pacientes (AU)

Cambios histopatológicos en el tejido gingival enfermo / Histopathological changes in the diseased gingival tissue

Se realizó un estudio descriptivo y transversal en pacientes que acudieron al Servicio de Periodoncia de la Clínica Estomatológica Provincial Docente de Santiago de Cuba, cuyo universo estuvo constituído por 22 pacientes necesitados de tratamiento quirúrgico. El objetivo de este trabajo fue el de determinar los cambios epiteliales y del corium más frecuentes y relacionarlos con los aspectos clínicos y el diagnóstico de la enfermedad periodontal, así como precisar la frecuencia de invasión bacteriana en el tejido gingival enfermo. En un formulario confeccionado al efecto, se anotaron: aspectos clínicos de la encía, diagnóstico clínico y cambios histopatológicos. Entre los principales hallazgos se encuentran que el cambio epitelial más frecuente resultó ser la acantosis, y que la invasión bacteriana se puso de manifiesto en el 13,6 por ciento de los pacientes