A broad-range PCR technique for the diagnosis of infective endocarditis

Abstract Infective endocarditis (IE) remains a severe and potentially fatal disease demanding sophisticated diagnostic strategies for detection of the causative microorganisms. The aim of the present study was to develop a broad-range 16S ribosomal RNA gene polymerase chain reaction in the routine diagnostic of IE for the early diagnosis of fatal disease. A broad-range PCR technique was selected and evaluated in terms of its efficiency in the diagnosis of endocarditis using 19 heart valves from patients undergoing cardiovascular surgeries at the Habib Bourguiba Hospital of Sfax, Tunisia, on the grounds of suspected IE. The results demonstrated the efficiency of this technique particularly in cases involving a limited number of bacteria since it helped to increase detection sensitivity. The technique proved to be efficient, particularly, in the bacteriological diagnosis of IE in contexts involving negative results from conventional culture methods and other contexts involving bacterial species that were not amenable to identification by phenotypic investigations. Indeed, the sequencing of the partial 16S ribosomal RNA gene revealed the presence of Bartonella henselae, Enterobacter sp., and Streptococcus pyogenes in three heart valves with the negative culture. It should be noted that the results obtained from the polymerase chain reaction-sequencing identification applied to the heart valve and the strain isolated from the same tissue were not consistent with the ones found by the conventional microbiological methods in the case of IE caused by Gemella morbillorum. In fact, the results from the molecular identification revealed the presence of Lactobacillus jensenii. Overall, the results have revealed that the proposed method is sensitive, reliable and might open promising opportunities for the early diagnosis of IE.

Microbiota periodontal y microorganismos aislados de válvulas cardiacas en pacientes sometidos a cirugía de reemplazo de válvulas en una clínica de Cali, Colombia / Periodontal microbiota and microorganisms isolated from heart valves in patients undergoing valve replacement surgery in a clinic in Cali, Colombia

Resumen Introducción. La periodontitis es una enfermedad infecciosa que afecta los tejidos de soporte del diente y se asocia con diferentes enfermedades sistémicas, incluida la enfermedad cardiovascular. Los estudios microbiológicos permiten detectar microorganismos a partir de muestras subgingivales y cardiovasculares. Objetivo. Describir la microbiota periodontal cultivable y la presencia de microorganismos en válvulas cardiacas de pacientes sometidos a cirugía de reemplazo valvular en una clínica de Cali. Materiales y métodos. Se analizaron 30 muestras subgingivales y de tejidos valvulares mediante cultivo en medio bifásico, agar de sangre con suplemento y agar tripticasa de soya con antibiótico. Las muestras de las válvulas se analizaron mediante reacción en cadena de la polimerasa (PCR) convencional. Resultados. Los patógenos periodontales aislados de bolsas periodontales fueron Fusobacterium ( 50 % ), Prevotella intermedia/nigrescens (40 %), Campilobacter rectus (40 %), Eikenella corrodens (36,7 %), bacilos entéricos Gram negativos (36,7 %), Porphyromonas gingivalis (33,3 %) y Eubacterium (33,3 %). Los agentes patógenos aislados de la válvula aórtica fueron Propionibacterium acnes (12 %), bacilos entéricos Gram negativos (8 %), Bacteroides merdae (4 %) y Clostridium bifermentans (4 %), y de la válvula mitral, P. acnes y Clostridium beijerinckii. La PCR convencional no arrojó resultados positivos para agentes patógenos orales y solo se detectó ADN bacteriano en dos muestras. Conclusiones. La microbiota periodontal de pacientes sometidos a cirugía de reemplazo valvular estaba conformada por especies Gram negativas que han sido relacionadas con infecciones en tejidos extraorales; sin embargo, no se encontraron agentes patógenos periodontales en los tejidos de las válvulas. Aunque hubo muestras de estos tejidos y subgingivales, positivas para bacilos entéricos Gram negativos, no es posible asegurar que tuvieran el mismo origen filogenético.

Abstract Introduction: Periodontitis is an infectious disease that affects the support tissue of the teeth and it is associated with different systemic diseases, including cardiovascular disease. Microbiological studies facilitate the detection of microorganisms from subgingival and cardiovascular samples. Objective: To describe the cultivable periodontal microbiota and the presence of microorganisms in heart valves from patients undergoing valve replacement surgery in a clinic in Cali. Materials and methods: We analyzed 30 subgingival and valvular tissue samples by means of twophase culture medium, supplemented blood agar and trypticase soy agar with antibiotics. Conventional PCR was performed on samples of valve tissue. Results: The periodontal pathogens isolated from periodontal pockets were: Fusobacterium nucleatum (50%), Prevotella intermedia/ nigrescens (40%), Campylobacter rectus (40%), Eikenella corrodens (36.7%), Gram negative enteric bacilli (36.7%), Porphyromonas gingivalis (33.3%), and Eubacterium spp. (33.3%). The pathogens isolated from the aortic valve were Propionibacterium acnes (12%), Gram negative enteric bacilli (8%), Bacteroides merdae (4%), and Clostridium bifermentans (4%), and from the mitral valve we isolated P. acnes and Clostridium beijerinckii. Conventional PCR did not return positive results for oral pathogens and bacterial DNA was detected only in two samples. Conclusions: Periodontal microbiota of patients undergoing surgery for heart valve replacement consisted of species of Gram-negative bacteria that have been associated with infections in extraoral tissues. However, there is no evidence of the presence of periodontal pathogens in valve tissue, because even though there were valve and subgingival samples positive for Gram-negative enteric bacilli, it is not possible to maintain they corresponded to the same phylogenetic origin.

Endocarditis Infecciosa: caracterización clínica de la enfermedad: revisión de casos de los últimos 5 años / Infective endocarditis: clinical experience in the last five years

Antecedentes: La Endocarditis infecciosa es una enfermedad grave y su incidencia ha aumentado en el último tiempo. En Chile faltan estudios observaciona-les que representen adecuadamente nuestra realidad local y nacional. Objetivo: Describir las características clínicas, ecográficas y bacteriológicas de los pacientes con Endocarditis Infecciosa del Hospital Carlos Van Buren de Valparaíso, entre los años 2012 y 2016. Métodos: Se realizó un análisis descriptivo de 35 pacientes con diagnóstico de Endocarditis infecciosa, recolectándose variables clínicas, imagenológicas y de laboratorio. Para describir los resultados se utilizó frecuencias, medianas y figuras. Resultados: Se observó un aumento de casos de Endocarditis infecciosa en los últimos dos años. Del total de pacientes con Endocarditis infecciosa, 28 casos (80%) eran de válvula nativa, siendo el compromiso más frecuente el de válvula aórtica. Se observó un aumento de las comorbilidades no cardíacas y de los casos provocados por el grupo Staphylococcus. Los síntomas más comunes fueron compromiso del estado general y fiebre, y las complicaciones más frecuentes fueron insuficiencia renal aguda, insuficiencia cardíaca aguda y fenómenos embólicos. Un total de 14 pacientes fallecieron producto de la infección, las complicaciones y cirugía. Conclusiones: Se realiza una caracterización actualizada de la enfermedad. Se necesitan más estudios con un mayor número de paciente para un mejor entendimiento de nuestra realidad.

Background: Infective endocarditis is a severe illness the incidence of which has increased over time. There are relatively few observational studies that adequately represent the characteristics of the disease in Chile. Aim: To describe clinical, echographic and bacteriological characteristics of patients with infective endocarditis at Carlos van Buren Hospital in Valparaíso, between 2012 and 2016. Methods: A descriptive analysis of 35 patients diagnosed with infective endocarditis was performed. Cinical, imaging findings and laboratory variables were collected. Relative frequencies, medians and figures were used to describe the results. Results: We observed an increase in cases of infective endocarditis in the last two years. Of all patients diagnosed with infective endocarditis, 80% had native valve involvement, more frequently at the aortic valve. There was an increase in non-cardiac comorbidities and Staphylococcus was identified as the main etiology. Weakness and fever were the most common symptoms, whereas more common complications were acute kidney and heart failure and embolic phenomena. A total of 14 patients died of infection, complications or after surgery surgery. Conclusions: An updated characterization of Infective endocarditis at a Chilean hospital is presen-ted. However, more studies are needed with a larger number of patients for a better characterization of the disease in our country.

Infective endocarditis involving an apparently structurally normal valve: new epidemiological trend?

Infective endocarditis (IE) has been increasingly diagnosed in patients without previously detected predisposing heart disease, but its clinical features have yet to be fully determined. A recent single-center study including echocardiographic images and surgical findings investigated the incidence of undiagnosed, clinically silent valvular or congenital heart diseases and healthcare-associated infective endocarditis (HAIE). The study confirmed that a large proportion of patients with IE have no previous history of heart disease. Analysis of underlying disease in these patients showed that undetected mitral valve prolapse was the most common disease, followed by an apparently structurally normal valve. The patients who developed IE of apparently structurally normal valves had different clinical characteristics and worse outcomes. IE involving a structurally normal valve was associated with both nosocomial and non-nosocomial HAIE, whereas community-acquired IE was more frequent than HAIE. The pathophysiologic mechanism involving the development of non-HAIE or community-acquired IE due to predominantly staphylococcal infection in an apparently structurally normal valve is not yet clearly understood. Structurally normal valves are not necessarily free of regurgitation or abnormal turbulence and, given the dynamic nature and fluctuating hemodynamic effects of conditions such as poorly controlled hypertension, end-stage renal disease, and sleep apnea, further investigation is necessary to evaluate the potential role of these diseases in the development of IE. An apparently normal-looking valve is associated with IE development in patients without previously recognized predisposing heart disease, warranting repartition of at-risk groups to achieve better clinical outcomes.

Endocarditis infecciosa activa: 152 casos / Active infective endocarditis: 152 cases

La endocarditis infecciosa es una enfermedad de baja incidencia que en las últimas décadas mostró modificaciones respecto de su presentación, posibilidad diagnóstica y tratamiento. A pesar de estos avances, la mortalidad hospitalaria sigue siendo muy elevada. Nuestro objetivo fue analizar las características de los pacientes con endocarditis infecciosa activa y su relación con la mortalidad hospitalaria a lo largo de 16 años. Se realizó un registro prospectivo entre 1994 y 2010 de pacientes ingresados con endocarditis. Se analizaron características clínicas, evolución y tratamiento y se registraron los eventos intrahospitalarios. Ingresaron 152 pacientes, 64.5% varones, edad 45 ± 16 años, las causas más frecuentes de cardiopatía de base fueron: congénita 32 (21%) y reumática 20 (13.2%). Los motivos de internación fueron síndrome febril 116 (76.3%) e insuficiencia cardíaca 61 (40.1%). Se identificó el agente infeccioso en 106 (69.7%) de los casos, el más frecuente fue Streptococcus viridans. El ecocardiograma mostró vegetaciones en 123 (80.9%) de los pacientes y 88 (57.8%) presentaron complicaciones durante su internación, siendo la más frecuente la insuficiencia cardíaca. Se indicó tratamiento quirúrgico en 96 (63.1%) de los casos, fundamentalmente por insuficiencia cardíaca en 66. La mortalidad hospitalaria global fue 46 (30.2%). El desarrollo de complicaciones en la internación, la indicación de cirugía y la presencia de insuficiencia cardíaca refractaria al tratamiento fueron predictores independientes de mortalidad hospitalaria, mientras que la presencia de vegetaciones resultó un predictor independiente de mejor supervivencia. La identificación temprana de estos predictores descriptos podría ayudar a mejorar los resultados.

Active infective endocarditis (IE) is a disease of low incidence that has showed changes in presentation, diagnosis and treatment options during the past decades. Despite these advances, mortality remains very high. Our goal was to analyze the characteristics of patients with active IE and their relationship with in-hospital mortality over 16 years. Between 1994 and 2010 we performed a prospective registry of 152 consecutive patients (64.5% male, age 45 ± 16 years) admitted with IE. Clinical characteristics, treatment and inpatient outcomes were analyzed. The most common causes of underlying heart disease were: congenital (21%) and rheumatic fever (13.2%). The reasons for hospitalization were fever (76.3%) and heart failure (40.1%). The infectious agent was identified in 69.7% of cases, and the most frequent was Streptococcus viridans. The echocardiogram showed vegetations in 80.9% of patients and 57.8% of them presented complications (the most frequent was heart failure) during hospitalization. Surgical treatment was indicated in 63.2% of cases, mainly due to heart failure. The overall hospital mortality was 30.2%. The presence of complications, requirement of surgical treatment and refractory heart failure were independent predictors of mortality whereas the single presence of vegetation showed better survival rate. The identification of these predictors could help to improve the outcomes in IE.

Presencia de ADN bacteriano en el tejido valvular de pacientes con cardiopatía reumática crónica / Presence of bacterial DNA in valvular tissue of patients with rheumatic heart disease

Background: Rheumatic heart disease (RHD) is a delayed consequence of a pharyngeal infection with Group A streptococcus (GAS), usually ascribed to a cross-reactive immune response to the host cardiac tissues. Acute rheumatic fever (ARF) and its ensuing valvular sequelae are thus considered the prototype of a post-infectious autoimmune disease, with no direct evidence of residual streptococcal antigen in diseased valvular tissues. However, recent studies concerning the antigenic specificity and clonality of intralesional lymphocytes have revealed oligoclonal expansions characteristic of an antigen specific response, that might be related to GAS. Aim: To search for bacterial DNA in valvular tissue from RHD patients and controls. Material and methods: We extracted DNA from surgically excised valve specimens from 15 RHD patients and 6 non RHD controls and tested for the presence of bacterial DNA by Polymerase Chain Reaction (PCR) with primers for 16S rRNA. Results: Eighty percent (12/15) of valve specimens from RHD patients were positive for bacterial DNA, as opposed to none of the valves (n =6) from non RHD controls. Conclusions: These results suggest that GAS might persist in valvular tissue in patients with ARF and contribute to the inflammatory scarring lesion that leads to cardiovascular sequelae.

Controle microbiológico em valvas cardíacas humanas / Microbiologyc control in human heart valves

OBJETIVO: Avaliar, sob o aspecto microbiológico, valvas processadas pelo Banco de Valvas Cardíacas Humanas da Irmandade da Santa Casa de Misericórdia de Curitiba, para serem utilizadas em cirurgias cardiovasculares. MÉTODOS: Foi avaliado o processamento de 1.671 valvas, no período de junho de 1999 a junho de 2004. Das valvas e soluções envolvidas no processo foram coletadas amostras e semeadas nos meios de cultura: meio líquido tioglicolato, caldo soja tripticaseína e caldo Sabouraud, com quatorze dias de incubação, utilizando a metodologia modificada baseada na Farmacopéia Brasileira 1998 e USP 1990 (United States Pharmacopeia). Nas amostras que apresentaram crescimento foram realizadas as identificações microbianas. RESULTADOS: Em um total de 1.671 amostras analisadas, 92 por cento foram consideradas próprias para utilização, sob o aspecto microbiológico, uma vez que não apresentaram contaminação microbiana. Somente 8 por cento não foram liberadas para uso clínico por motivo de contaminação em alguma etapa do processamento da valva. CONCLUSÃO: Analisando os resultados, observou-se a importância do controle microbiológico em enxertos humanos, evitando a utilização de valvas com contaminação microbiológica em pacientes submetidos à cirurgia cardiovascular.

OBJECTIVE: To evaluate, from microbiological point of view, the valves processed by Human Heart Valve Bank of Santa Casa de Misericórdia of Curitiba for use in cardiovascular surgeries. METHODS: The processing of 1,671 valves, accomplished within the period of time between July 1999 and June 2004, was evaluated. Out of the valves and the solutions involved in the process, samples were collected and spread in culture mediums, such as fluid thioglycollate medium, tryptic soy broth and Sabouraud broth, for incubation during 14 days, using a modified methodology based on the Farmacopéia Brasileira 1988 (Brazilian Pharmacopeia) and USP 1990 (United States Pharmacopeia). The samples in which growing was observed were submitted to microbian identification. RESULTS: In a set of 1,671 samples, 92 percent were considered proper for use under microbiological point of view, since they did not display microbian contamination. The remaining 8 percent were rejected for clinical use because of contamination in some stage of the valve processing. CONCLUSION: From the Analysis of the results, it was observed the importance of microbiological control in human grafts, in order to avoid using microbiologically contaminated valves in patients submitted to cardiovascular surgery.

Recomendaciones para el estudio microbiológico de tejidos preservados para implantes / Recommendations for the microbiological study of tissues preserved for implants

El uso de los implantes de tejido en medicina ha mejorado el pronóstico y/o la calidad de vida de muchas enfermedades, sin embargo, su utilización no está exenta de riesgos para el receptor. Es por esto que la cuidadosa selección del donante vivo o cadáver y el control microbiológico de los tejidos antes de su utilización son procesos muy importantes para asegurar la calidad del procedimiento. La contaminación de los tejidos puede ser intrínseca (por una infección no diagnosticada en el donante) o extrínseca (una vez que el tejido ya ha sido procurado). Estas recomendaciones se refieren al control microbiológico de las contaminaciones extrínsecas, ya sea durante la manipulación, durante la obtención de los tejidos o durante el procesamiento para la preservación de ellos. Se analizan los implantes de uso más frecuente en nuestro país (válvulas cardíacas, tejido óseo, piel, células progenitoras hematopoyéticas y córnea) y se describe la metodología según datos existentes en la literatura y de procedimientos adaptados de centros foráneos. Dado que ningún implante es absolutamente estéril es muy importante analizar también las recomendaciones frente a cultivos contaminados. Los autores esperamos que este documento sea de utilidad y que en un futuro próximo, podamos exhibir cifras nacionales multicéntricas con metodologías comparables.

Patogénesis de la endocarditis infecciosa / Pathogenesis of infectious endocarditis

Se discuten los mecanismos moleculares que llevan a la formación de las lesiones más características de la Endocarditis Infecciosa, en especial, la formación de vegetaciones y los diferentes mediadores involucrados provenientes de los gérmenes causales.

Pathogenesis of infective endocarditis.

The present knowledge of epidemiology, microbiology and pathogenesis of infective endocarditis in both native valve and prosthetic valve endocarditis is described. An attempt has been made to discuss early events in its pathogenesis. This understanding may help in the prevention and management strategies.

Endocarditis infecciosa: análisis de 261 casos y resultados del tratamiento con un enfoque multidisciplinario / Infectious endocarditis: short and long term results in 261 cases managed by a multidisciplinary approach

Background: Early diagnosis, an effective treatment and prompt recognition of complications are essential to improve the prognosis of infective endocarditis (IE) Aim: To report the results of a multidisciplinary approach to diagnosis and management of patients with IE at the Universidad Cat-lica de Chile Hospital. Patients and methods: The clinical history, diagnosis, treatment and outcome of 261 episodes (Duke criteria) of IE admitted between January 1980 and January 1999 were analyzed. These included 185 episodes of native, 73 of prosthetic valve and 3 of nonvalvular IE. Results: Sixty nine percent of patients were men and the mean age was 49 ñ 16 years. Seventy five percent had a definite diagnosis of IE (Duke). S. viridans, staphylococci and enterococci together constituted 85 percent of the isolated bacterial strains. Twenty seven had culture-negative IE, related to a high incidence of antibiotic therapy prior to diagnosis. Transesophageal echocardiography was performed in 102 cases and it detected vegetations in 91 percent of aortic and 96 percent of mitral IE, rupture or prosthesis dehiscence in 67 percent of aortic and 52 percent of mitral IE and abscesses in 51 percent of aortic and 15 percent of mitral IE. Fifty one percent developed heart failure and 34 percent had embolic events. S. aureus IE was associated to a higher incidence of embolic events, complications which contraindicated surgery and increased mortality rate (27 percent). Of all patients, 40 percent were treated exclusively with antibiotics, 52 percent were operated on and 8 percent had surgical indication but were nonoperable because of serious complications. The overall mortality was 16.3 percent: 13 percent in the medical, 9 percent in the surgical and 81 percent in the non-operable groups. The type of treatment and mortality rates did not differ between IE of native valves and prosthetic valves. Long term follow up showed survival rates of 73 percent at 5 years and 66 percent at 10 years. Conclusion: A multidisciplinary approach may be very helpful to improve the prognosis of IE

Endocarditis infecciosa valvular protésica / Prosthetic valve endocarditis

Se analizan las características clínicas, bacteriológicas, complicaciones, tratamiento y mortalidad hospitalaria de 25 endocarditis en válvulas protésicas (22 mecánicas y 3 biológicas) incluídas en el estudio E.I.R.A. De ellas, 7 eran endocarditis protésicas precoces y 18 tardías. La insuficiencia cardíaca grave se presentó como la complicación más frecuente en las precoces con respecto a las tardías (p= 0,01), mientras que las embolias no presentaron esa diferencia. La microbiología mostró al grupo Staphylococcus como el más frecuente en las precoces y al Streptococcus en las tardías. La mortalidad de las tardías fue elevada