The Pathology of Severe Dengue in Multiple Organs of Human Fatal Cases: Histopathology, Ultrastructure and Virus Replication

The Pathology of Severe Dengue in Multiple Organs of Human Fatal Cases: Histopathology, Ultrastructure and Virus Replication Tiago F. Póvoa1, Ada M. B. Alves1, Carlos A. B. Oliveira2, Gerard J. Nuovo3, Vera L. A. Chagas4, Marciano V. Paes1* 1 Laboratório de Biotecnologia e Fisiologia de Infecções Virais, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil, 2 Hospital Universitário Gaffrée Guinle, Departamento de Anatomia Patológica, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil, 3 University Comprehensive Cancer Center, Columbus, Ohio, United States of America, 4 Hospital Universitário Clementino Fraga Filho, Departamento de Anatomia Patológica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Abstract Dengue is a public health problem, with several gaps in understanding its pathogenesis. Studies based on human fatal cases are extremely important and may clarify some of these gaps. In this work, we analyzed lesions in different organs of four dengue fatal cases, occurred in Brazil. Tissues were prepared for visualization in optical and electron microscopy, with damages quantification. As expected, we observed in all studied organ lesions characteristic of severe dengue, such as hemorrhage and edema, although other injuries were also detected. Cases presented necrotic areas in the liver and diffuse macro and microsteatosis, which were more accentuated in case 1, who also had obesity. The lung was the most affected organ, with hyaline membrane formation associated with mononuclear infiltrates in patients with pre-existing diseases such as diabetes and obesity (cases 1 and 2, respectively). These cases had also extensive acute tubular necrosis in the kidney. Infection induced destruction of cardiac fibers in most cases, with absence of nucleus and loss of striations, suggesting myocarditis. Spleens revealed significant destruction of the germinal centers and atrophy of lymphoid follicles, which may be associated to decrease of T cell number. Circulatory disturbs were reinforced by the presence of megakaryocytes in alveolar spaces, thrombus formation in glomerular capillaries and loss of endothelium in several tissues. Besides histopathological and ultrastructural observations, virus replication were investigated by detection of dengue antigens, especially the non-structural 3 protein (NS3), and confirmed by the presence of virus RNA negative strand (in situ hybridization), with second staining for identification of some cells. Results showed that dengue had broader tropism comparing to what was described before in literature, replicating in hepatocytes, type II pneumocytes and cardiac fibers, as well as in resident and circulating monocytes/macrophages and endothelial cells. Citation: Póvoa TF, Alves AMB, Oliveira CAB, Nuovo GJ, Chagas VLA, et al. (2014) The Pathology of Severe Dengue in Multiple Organs of Human Fatal Cases: Histopathology, Ultrastructure and Virus Replication. PLoS ONE 9(4): e83386. doi:10.1371/journal.pone.0083386 Editor: Xia Jin, University of Rochester, United States of America Received May 3, 2013; Accepted February 17, 2014; Published April 15, 2014 Copyright: ß 2014 Póvoa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding provided by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq); Programa de Excelência em Pesquisa (PROEP/CNPq/FIOCRUZ); Programa de Apoio a Núcleos de Excelencia (PRONEX)–CNPq/FAPERJ; Instituto Nacional de Ciência e Tecnologia de Vacinas (INCTV)/CNPq grants. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: age, co-morbidities, genetic predisposition and immune conditions of the patient, as well as genetic variations of viral strains, may also contribute for the occurrence of DHF [8–10]. Severe dengue disease is characterized by circulatory damages, associated in most cases with hepatic dysfunctions [11–13]. These injuries may be a direct consequence of the virus presence and/or resulted by an exacerbation of the immune response after infection [14,15]. Overall, in vivo studies regarding DENV infection and its pathogenesis are limited by the lack of an experimental animal model able to mimic the full spectrum of the disease as observed in humans [16]. Therefore, there are still several gaps in understanding the pathogenesis of dengue. On the other hand, autopsy studies based on human dengue cases are extremely important and may clarified some of these gaps, pointing out for example how and which tissues are affected during the disease. Most of histopathological reports with dengue human fatal cases indicate Introduction Dengue infection is the most prevalent arthropod-borne viral disease in subtropical and tropical regions of the world. The dengue virus (DENV) belongs to the Flaviviridae family and consists of four antigenically distinct serotypes (DENV1-4). The infection can result in a broad spectrum of effects, including acute febrile illness, the dengue fever (DF), which may progress to severe forms such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), with changes in hemostasis and vascular permeability [1,2]. Several studies indicate that the occurrence of secondary infection with a heterologous serotype increase the risk of developing DHF [3,4]. Therefore, in areas where multiples DENV serotypes circulate, such as in Brazil, sequential infections may occur, which lead to the increase in the number of severe dengue cases [5–7]. Moreover, other risk factors, such as ethnicity, PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e83386 The Pathology of Dengue in Human Fatal Cases that the liver, spleen and lymph nodes are target organs of infection [17–19]. Besides the occurrence of hemorrhage and edema in the liver of dengue fatal cases, histopathological analysis also reported damages caused by metabolic alterations and/or inflammatory reactions, such as the presence of steatosis, areas with infiltrated cells and necrosis and hyperplasia and destruction of Kupffer cells [17,18,20–22]. Additionally, other studies showed several lesions in spleen tissues, such as interstitial edema, vascular congestion, splenic rupture and bleeding [17,18,23]. However, recently, atypical clinical manifestations of dengue have been reported, involving the kidney, lung, heart and central nervous system, which were also corroborated by histopathological findings revealing several areas with hemorrhage, edema and inflammatory infiltrates in these organs [24–28]. In addition (...truncated)