Calcificações vasculares nos doentes em diálise : elo de ligação entre doença óssea e doença vascular

RESUMO: A presente dissertação para tese de doutoramento apresenta o desenvolvimento e a validação de um método simples e original para o diagnóstico de calcificações vasculares em doentes em diálise, utilizando um score semiquantitativo criado por nós e obtido em RX simples da bacia e das mãos, denominado score de calcifi cação vascular simples. Demonstramos que este score vascular simples é preditor de risco cardiovascular nos doentes em diálise. O score de calcificação vascular simples associou-se ainda à baixa densidade mineral óssea avaliada por dual energy X -ray absortiometry (DXA) no colo do fémur. Verifi camos igualmente que, em doentes em diálise, as calcifi cações coronárias quantifi cadas pelo score de Agatston e o score de calcifi cação vascular simples se associaram a um menor volume ósseo avaliado em biopsias ósseas. Estes trabalhos corroboram a hipótese da existência de um elo de ligação entre a doença óssea e a doença vascular nos doentes em diálise, e um dos elementos que contribuem para este elo de ligação podem ser as calcificações vasculares. Este score de calcificação vascular simples avalia calcifi cações em artérias de grande, médio e pequeno calibre, e inclui os dois padrões radiológicos de calcificação: calcificação linear, associada à calcifi cação da camada média da parede arterial, e calcificação irregular, associada à calcifi cação da camada íntima arterial1. Nos diferentes trabalhos por nós publicados demonstramos que as calcificações vasculares avaliadas por este método simples e barato permitem a identificação de indivíduos com elevado risco cardiovascular. Este score vascular associa -se a maior risco de mortalidade cardiovascular2, de mortalidade de causa global3, de internamentos cardiovasculares2, de doença ardiovascular2, de doença arterial periférica2,4,de calcifi cações valvulares5 e de rigidez arterial3. As guidelines KDIGO (Kidney disease: improving global outcomes), publicadas em 2009,sugerem que os doentes renais crónicos nos estadios 3 a 5, com calcificações vasculares e valvulares, devem ser considerados como apresentando o mais elevado risco cardiovascular6. A elevada mortalidade dos doentes renais crónicos não é totalmente explicada pelos fatores de risco tradicionais7. A organização KDIGO defende, desde 2006, a hipótese da existência de um elo de ligação entre a doença óssea e a doença vascular8. Esta ligação pode ser explicada pelas alterações do metabolismo mineral e ósseo e pela sua interação com as calcificações vasculares. Verificamos, nos nossos trabalhos, uma associação entre calcifi cações vasculares e doença óssea. O baixo volume ósseo diagnosticado por análise histomorfométrica de biopsias ósseas foi preditor de maior risco de calcificações vasculares avaliadas pelo score de calcifi cação vascular simples (dados apresentados nesta dissertação, no capítulo 6) e pelo score coronário de Agatston num grupo de doentes em diálise9. A contribuição original deste artigo9 foi considerada merecedora de um editorial feito pelo Dr. Gérard London10, investigador líder na área da calcificação vascular dos doentes renais crónicos e actual Presidente da EDTA (European Dialysis and Transplantation Association). Fomos também os primeiros a descrever uma associação independente e inversa entre a densidade mineral avaliada no colo do fémur por DXA (dual energy X -ray absortiometry) com calcificações vasculares avaliadas pelo score de calcificação vascular simples, com rigidez arterial avaliada por velocidade de onda de pulsocarotidofemoral e com doença arterial periférica diagnosticada por critérios clínicos11. Fomos igualmente os primeiros a mostrar uma correlação signifi cativa entre a densidade mineral óssea avaliada por DXA no colo do fémur, mas não na coluna lombar, com a espessura cortical avaliada por análise histomorfométrica em biopsia óssea12. O nosso estudo atribui pela primeira vez à DXA um papel no diagnóstico de porosidade cortical nos doentes em diálise. A utilidade da avaliação diferencial da densidade mineral óssea cortical e trabecular necessita ainda de ser confirmada em estudos prospectivos. Este achado inovador do nosso estudo foi mencionado pela ERBP (European Renal Best Practice) no comentário feito à posição da KDIGO que considera ser reduzida a utilidade da densidade mineral óssea nos doentes em diálise13. Dois dos trabalhos incluídos nesta dissertação foram referenciados nas guidelines KDIGO 2009 para avaliar a prevalência das calcificações vasculares (KDIGO 2009: Tabela suplementar 10, Fig. 3.6) e para validar a associação entre calcificações vasculares e mortalidade cardiovascular (KDIGO 2009: Tabela suplementar 12, Fig. 3.7)6. A inclusão destes nossos dois estudos nas referências destas guidelines, que utilizaram o exigente sistema GRADE (Grades of recommendation, assessment, development, and evaluation) na classificação e selecção dos estudos, valida o interesse científico dos nossos trabalhos. O diagnóstico de calcificações vasculares tem um interesse prático para os doentes renais crónicos. A presença de calcifi cações vasculares é um sinal de alerta para a existência de um elevado risco cardiovascular, e esta informação pode ser utilizada para modificar a terapêutica nestes doentes6. Diferentes métodos podem ser usados para diagnosticar calcificações vasculares nos doentes em diálise14,15. O score de calcificação vascular simples tem a vantagem da simplicidade e de poder ser facilmente interpretado pelo nefrologista, sem necessidade de um radiologista. A reprodutibilidade deste score já foi demonstrada por diferentes grupos em estudos nacionais e internacionais16-24. Nestes estudos foi demonstrado que as calcifi cações vasculares avaliadas pelo método criado por nós são preditoras de maior risco de eventos cardiovasculares16, de amputações dos membros inferiores17, de velocidade de onda de pulso18,19, de calcificações corneanas e conjuntivais20 e de calcifi cações coronárias21. Também foi demonstrada uma associação inversa entre o score de calcificação vascular simples com os níveis séricos de PTH21, com os níveis de 25(OH)vitamina D 22,23 e com os níveis de fetuína A19,24. Todos estes estudos, realizados por diferentes grupos, que utilizaram o score de calcificação vascular simples na sua metodologia, comprovam a facilidade de utilização deste score e a concordância de resultados atestam a sua reprodutibilidade e a utilidade na avaliação dos doentes renais crónicos. ---------------------------ABSTRACT: This thesis presents the development and validation of a simple and original method to identify vascular calcifications in dialysis patients, using a semi -quantitative score that we have created and that is obtained in plain X -ray of pelvis and hands. This score was named in different publications as “simple vascular calcifi cation score”. We have demonstrated that this score is a predictor of higher cardiovascular risk in dialysis patients. The simple vascular calcification score was also associated with lower mineral bone density evaluated by DXA in femoral neck. In hemodialysis patients coronary calcifications evaluated by the coronary Agatston score and by the simple vascular calcification score were associated with lower bone volume analysed in bone biopsies. These studies corroborate the hypothesis of the existence of a link between bone disease and vascular disease in dialysis patients and one of the elements of this link may be vascular calcifications. This simple vascular calcification score identifi es calcifications in large, medium and small calibre arteries and includes the two radiological patterns of arterial calcifi cation: linear calcification which has been associated with the calcifi cation of the media layer of the arterial wall and irregular and patchy calcification which has been associated with the calcifi cation of the intima layer of the arterial wall1. In the several studies that we have published we have demonstrated that vascular calcifications evaluated by this simple and inexpensive method allow the identification of patients with high cardiovascular risk. This simple vascular calcification score is an independent predictor of cardiovascular mortality2, all -cause mortality3, cardiovascular hospitalizations2, cardiovascular disease2, peripheral artery disease2,4, valvular calcifi cations5 and arterial stiffness3.KDIGO (Kidney Disease: Improving Global Outcomes) guidelines published in 2009 suggest that chronic kidney disease patients in stages 3 to 5, with vascular and valvular calcifications should be considered to be at the highest cardiovascular risk6. The high mortality of chronic kidney disease patients is not completely explained by the traditional risk factors7 and KDIGO group supports, since 2006, the hypothesis of the existence of a link between bone disease and vascular disease8.This link may be explained by the alterations of the bone and mineral metabolism and their interaction with development and progression of vascular calcifications. We have also verifi ed in our studies the existence of an association between vascular calcifications and bone disease. Low bone volume diagnosed by histomorphometric analysis of bone biopsies, in a group of dialysis patients, was independently associated with the simple vascular calcification score (data presented in this thesis,chapter 6) and with coronary calcifications evaluated by the Agatston score9. The original contribution of this article published in CJASN9 deserved a commentary in an Editorial written by Prof. Gérard London10 leader investigator in this area and current EDTA (European Dialysis and Transplantation Association) President. We were also the fi rst group to describe an independent and inverse association between bone mineral density evaluated in the femoral neck by DXA (dual energy X -ray absortiometry) with vascular calcifications evaluated by the simple vascular calcification score, with arterial stiffness evaluated by carotid-femoral pulse wave velocity and with peripheral artery disease diagnosed by clinical criteria11. We were also the first group to demonstrate a significant correlation between bone mineral density evaluated by DXA in femoral neck but not in lumbar spine, with cortical thickness evaluated by histomorphometric analysis of bone biopsy12. Our study has attributed to DXA, for the first time, a role in the diagnosis of cortical porosity in dialysis patients. The clinical utility of the differential evaluation of bone mineral density in cortical or trabecular bone needs, however, to be confi rmed in prospective studies. This original fi nding of our study was mentioned by ERBP (European Renal Best Practice) commenting the KDIGO position in relation with the reduced utility of bone mineral density evaluation in dialysis patients13. Two of the studies included in this thesis have been integrated in a group of studies selected as references by the KDIGO guidelines published in 2009 to evaluate the prevalence of vascular calcifications in CKD patients (KDIGO 2009: Supplementary Table 10, Fig. 3.6) and to corroborate the association between vascular calcifications and cardiovascular mortality (KDIGO 2009: Supplementary Table 12, Fig. 3.7)6. The inclusion of both studies as references in the KDIGO guidelines that have used the exigent GRADE system (Grades of Recommendation, Assessment, Development, and Evaluation) in the classifi cation and selection of studies, validates the scientifi c value of our studies. The diagnosis of vascular calcifi cations has a practical interest for chronic kidney disease patients. The presence of vascular calcifications is an alert sign to the existence of a high cardiovascular risk and this information may be used to modify the treatment of these patients6. Different methods may be used to detect the presence of vascular calcifications in dialysis patients14,15. The simple vascular calcifi cation score has the advantage of being simple, inexpensive and easily evaluated by the Nephrologist without the need for a Radiologist interpretation. The reproducibility of this method has already been demonstrated by other groups in national and international studies16 -24. It was demonstrated in those studies that vascular calcifi cations evaluated by the method created by us, predict higher risk of cardiovascular events16, higher risk of lower limbs amputations17, higher pulse wave velocity18,19, corneal and conjuntival calcifi cations 20 and coronary calcifi cations21. A negative association between the simple vascular calcification score and PTH levels21, 25(OH) vitamin D levels22,23 and Fetuin A levels19,24 has also been demonstrated. All these studies performed by different groups that have used the simple vascular calcifi cation score in their methods demonstrate that this score is simple, useful and reproducible in the evaluation of chronic kidney disease patients simple, useful and reproducible in the evaluation of chronic kidney disease patients.

Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Dissertation to obtain a Master Degree in Biotechnology

UNA virus: first report of human infection in Argentina

Una virus (UNAV), Togaviridae family, is widely distributed in South America, where infections have been detected in mosquitoes and vertebrate hosts (humans, birds and horses). We analyzed human sera from Córdoba inhabitants aged 44 to 89 years and using a neutralization test, we found a prevalence of UNAV antibodies of 3.8% (3/79). The low titers detected suggest past infections probably acquired in rural areas of the Province of Córdoba (central Argentina). None sera were found positive for MAYV neutralizing antibodies. This is the first report of human infections by UNAV in Argentina.

Fonsecaea pedrosoi cerebral phaeohyphomycosis ("chromoblastomycosis"): first human culture-proven case reported in Brazil

Cerebral phaeohyphomycosis ("chromoblastomycosis") is a rare intracranial lesion. We report the first human culture-proven case of brain abscesses due to Fonsecaea pedrosoi in Brazil. The patient, a 28 year-old immunocompetent white male, had ocular manifestations and a hypertensive intracranial syndrome. Magnetic resonance imaging (MRI) of the brain revealed a main tumoral mass involving the right temporo-occipital area and another smaller apparently healed lesion at the left occipital lobe. A cerebral biopsy was performed and the pathological report was cerebral chromoblastomycosis. The main lesion was enucleated surgically and culture of the necrotic and suppurative mass grew a fungus identified as Fonsecaea pedrosoi. The patient had received a knife wound sixteen years prior to his hospitalization and, more recently, manifested a pulmonary granulomatous lesion in the right lung with a single non-pigmented form of a fungus present. It was speculated that the fungus might have gained entrance to the host through the skin lesion, although a primary respiratory lesion was not excluded. The patient was discharged from the hospital still with ocular manifestations and on antimycotic therapy and was followed for eight months without disease recurrence. Few months after he had complications of the previous neuro-surgery and died. A complete autopsy was performed and no residual fungal disease was found.

First report of a human case of polycystic echinococcosis due to Echinococcus vogeli from neotropical area of Peru, South America

We report a human case of polycystic hidatidosis due to Echinococcus vogeli from Contamana (Department of Loreto) village located in the central jungle of Peru. The patient is a 44 year-old lady, teacher, who carried a painless liver mass since a year ago. She was submitted to abdominal surgery and the liver mass was removed and showed multiple cysts containing colorless liquid as is showed in the polycystic hidatidosis. The morphology and measure of the hooks obtained from the liquid contained in the cysts are from Echinococcus vogeli. It is the first report of this parasitism in Perú.

Mediastinal histoplasmosis: report of the first two Brazilian cases of mediastinal granuloma

This report documents the first two Brazilian cases of mediastinal granuloma due to histoplasmosis, presenting selected aspects on the diagnosis. Tissue samples revealing histoplasmosis were obtained from each of the patients by mediastinoscopy and thoracotomy. In the second patient, a subcarinal calcified mass eroded into the bronchial tree, leading to secondary bilateral aspiration pneumonitis one week after thoracotomy. Although rare, histoplasmosis should be included in the differential diagnosis of mediastinal granuloma, specially if there are calcifications greater than 10 mm in dimension.

First record of Desmodus rotundus in urban area from the city of Olinda, Pernambuco, Northeastern Brazil: a case report

The objective of this report is to describe the first record of Desmodus rotundus in urban area from the city of Olinda, Pernambuco State, Northeastern Brazil, and to draw attention to the possible risk of rabies transmission in this place. After the complaint of a dog owner who observed three bats attacking his dog, images registering attacks of D. rotundus were captured with a video camera. From 09:00 p.m. on 13 February 2004 to 04:00 a.m. of the next day, a high frequency of haematophagic activity and the presence of several bites on the dog's body were observed. This finding represents a serious risk to public health. Thus, it is necessary to further study the bat fauna, with special attention to their feeding behaviour in this place, in order to better know their biology and to adopt pertinent control measures. This is, to our knowledge, the first record of D. rotundus in urban area of Olinda.

Cerebral aspergillosis due to Aspergillus fumigatus in AIDS patient: first culture - proven case reported in Brazil

Cerebral aspergillosis is a rare cause of brain expansive lesion in AIDS patients. We report the first culture-proven case of brain abscess due to Aspergillus fumigatus in a Brazilian AIDS patient. The patient, a 26 year-old male with human immunodeficiency virus (HIV) infection and history of pulmonary tuberculosis and cerebral toxoplasmosis, had fever, cough, dyspnea, and two episodes of seizures. The brain computerized tomography (CT) showed a bi-parietal and parasagittal hypodense lesion with peripheral enhancement, and significant mass effect. There was started anti-Toxoplasma treatment. Three weeks later, the patient presented mental confusion, and a new brain CT evidenced increase in the lesion. He underwent brain biopsy, draining 10 mL of purulent material. The direct mycological examination revealed septated and hyaline hyphae. There was started amphotericin B deoxycholate. The culture of the material demonstrated presence of the Aspergillus fumigatus. The following two months, the patient was submitted to three surgeries, with insertion of drainage catheter and administration of amphotericin B intralesional. Three months after hospital admission, his neurological condition suffered discrete changes. However, he died due to intrahospital pneumonia. Brain abscess caused by Aspergillus fumigatus must be considered in the differential diagnosis of the brain expansive lesions in AIDS patients in Brazil.

St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

In Portugal (1912): Aubrey Bell's depiction of Portuguese society under the First Republic

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Línguas, Culturas e Literaturas Modernas

Listeria monocytogenes peritonitis in cirrhotic patients: first description in Brazil

Two cases of spontaneous bacterial peritonitis (SBP) caused by Listeria monocytogenes in cirrhotic patients are reported. In one of the cases, the microorganism was isolated from pleural effusion and ascites. SBP is a serious and common complication of patients with ascites caused by hepatic cirrhosis and the culture of the ascitic fluid is an important tool for the diagnosis and for the more appropriate treatment. Although a third generation cephalosporin has usually been employed for empiric treatment of SBP, it does not provide adequate coverage against Listeria spp. In such cases the use of ampicillin (with or without sulbactam) or sulfamethoxazole-trimethoprim is recommended. The last one is used for secondary prophylaxis, instead of norfloxacin. To summarize, Listeria monocytogenes infection is a rare cause of SBP, whose treatment should be specific for the bacteria.

Human infection by Angiostrongylus costaricensis in Venezuela: first report of a confirmed case

A proven case of human infection caused by Angiostrongylus costaricensis is reported for the first time in Venezuela. The patient was a 57-year-old female surgically operated because of signs of peritonitis with a palpable mass at the lower right quadrant of the abdomen. WBC count reported 16,600 cells/mm³, with 46% eosinophils. The tumoral aspect of ileocolic area and peritoneal lymph nodes prompted the resection of a large area of the terminal ileum, cecum, part of the ascending colon and a small part of the jejunum, where a small lesion was found. The pathology showed thickened areas of the intestinal wall with areas of hemorrhage and a perforation of the cecum. Histology showed intense eosinophil infiltration of the whole intestinal wall, granulomas with giant cells and eosinophils. Some of the granuloma surrounded round or oval eggs with content characterized by a large empty area, cells or embryo in the center, and sometimes nematode larvae. A cross section of an adult nematode worm was observed inside a branch of mesenteric artery. The intestinal affected area, the characteristics of the lesions, the presence of eggs in the submucosa with nematode larvae inside, and the observation of a nematode inside a mesenteric artery, makes sufficient criteria for the diagnosis of an infection by Angiostrongylus costaricensis.

First record of Anopheles (Anopheles) costai Fonseca & Ramos, 1939 in Espírito Santo State, Brazil

Field collections of immatures and adults of Culicidae were carried out in the Rio Doce Valley, Espírito Santo State, aiming to increase knowledge on the anopheline species of that region. Considering all Culicidae species collected, among other anopheline it is noteworthy the presence of Anopheles darlingi, An. oswaldoi and An. costai. Regarding to An. darlingi, responsible for malaria outbreak in the low lands in that state, it was expected to confirm its presence in the area. The register of specimens of An. costai in the field collections is relevant, increasing the geographical distribution of the species and representing better knowledge of the Series Arribalzagia of the subgenus Anopheles.

First report of rabies in vampire bats (Desmodus rotundus) in an urban area, Ubatuba, São Paulo state, Brazil

The purpose of this report is to record the first case of a hematophagous bat (Desmodus rotundus) infected with rabies virus in an urban area in Brazil. To the authors' knowledge, this is the first such case in Latin America. After discovering a bat in his garden at 10 o'clock in the morning, a resident of Ubatuba municipality asked the Zoonosis Control Center team to visit his home. The animal was caught alive on the same day and sent to the Pasteur Institute laboratory, where it was identified as a Desmodus rotundus specimen. Standard tests for rabies diagnosis were carried out (direct immunofluorescence and viral isolation), and the results were positive. The presence of different species of (primarily insectivorous) bats in urban areas represents a serious public health problem. This case, however, is indicative of a much greater risk because the species in question has hematophagous habits, what means this animals has a low energy reserves and, therefore, its need to feed daily.