PATHOLOGY OF EBOLA VIRUS INFECTION

So few specimens of tissue from fatal cases of Ebola virus disease have been available for pathologic study that no description can be considered representative, and any analysis of pathogenetic  mechanisms should be recognized as speculative. No report of gross pathologic findings is available and specimens for histopathologic study have consisted only of liver tissue from three cases in Zaire (sent to CDC by the WHO Field Team), and liver, spleen, and kidney tissues from two cases in the Sudan (sent to CDC by Dr. D.S. Ridley, Hospital for Tropical Diseases, London, and Dr. D.I.H. Simpson, London School of Hygiene and Tropical Medicine). Clinical pathology information will bepresented by other contributors to this Colloquium.

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Because of this paucity of tissue and lack of information of Ebola virus pathology it will be necessary to draw upon findings from Marburg virus studies carried out after the 1967 and 1975 episodes. Findings from 1967 primarily derive from the papers by P. Gedigk, H. Bechtelsheimer, G.Korb, and H. Jacob 1,2,3 ; pathologic findings from the single fatal case in South Africa in 1975 are not formally available but some observations are included here based upon a set of histologic slides sent to CDC by Professor J.H.S. Gear and his 4 colleagues of the South African Institute of Medical Research, Johannesburg (4).Ebola Virus InfectionThe three liver specimens from confirmed Ebola virus disease cases in Zaire were remarkably similar. There was fatty change and necrosis of hepatocytes and Kupffer cells (Figures 1,2). This necrosis was focally distributed throughout lobules, in some cases involving single cells and in other cases extending from central veins to lobular peripheries (Figures 3,4). The sequence of hepatocyte necrosis involved an initial cytoplasmic eosinophilia, then a shrinking, darkening and dissolution of nuclei (nucleoclasia). Intact cells with hyalin ized cytoplasm and ghostlike nuclei apparently remained in place for some time; but finally rarifaction, swelling and cytolysis occurred, leaving large amounts of karyorrhectic debris in situ. Considering the extent of this necrosis, there was remarkably little  inflammatory infiltration into sinusoids. There were large numbers of hepatocyte mitoses, but often binucleate cells underwent the same necrotic changes as they were entrapped by expanding foci of infection.

Extraordinary large and vivid intracytopl asmic eosinophilic inclusion bodies were present in hepatocytes in two of the three Zaire cases; further study showed that these represented an extreme and that large numbers of smaller and less distinct inclusions were present in all three cases (Figures5,6). Because smaller inclusions had indistinct margins and an eosinophilic color matching the early hyaline change of the cytoplasm of infected hepatocytes, they could only be identified with certainty in rather normal cells. Inclusion body stains helped in this identification, but as in most acute hepatocellular infections, the presence of large numbers of Councilman like bodies in areas of necrosis was a complicating factor. This matter has practical importance since histologic identification of Marburg/Ebola virus inclusions, in the presence of Councilman like bodies, could have presumptive diagnostic value in laboratories where virus isolation or immunofluorescent methods are not available.

The identity of the virus inclusions in the three Ebola cases was confirmed by thin section electron microscopy of formalin fixed liver tissue (Figure 7). Although preservation was poor, the inclusions found within the cytoplasm of hepatocytes were clearly made up of massed tubules which were identical to the internal constituent (? the nucleocapsid) of virus particles (Figure 8). These structures were indistinguishable from those found in Vero cell cultures infected with Ebola or Marburg viruses; similarly they were indistinguishable from hepatocyte inclusions in huma n, monkey and guinea pig(5,6,7) infections caused by Marburg virus . In the same three Zaire liver speci mens, large number of virus particles were found in all of the extracellular spaces (sinusoids, spaces of Disse and areas of necrosis); these were indistinguishable from Ebola virus particles in cell culture and Marburg virus particles in cell culture and in vivo (Figure 9). No Aiver necrosis was evident in the very small tissue specimens collected from fatal cases of hemorrhagic fever in the Sudan. There was some necrosis and calcification in tubules and glomerular tufts of kidney and there were necrotic cells in the spleen specimens. However, because no information was received about the course of disease nor laboratory

confirmation of the diagnosis, it is prudent to put off interpretation of this exceptional sparing of the liver. Marburg Virus Infection Comparison of the liver lesions of the Zaire Ebola cases with those of Marburg infections in 1967 and 1975 indicates precise similarities. At this point , further comparisons are impossible, but a brief review of Marburg disease pathology may be of value. In the fatal Marburg virus infection in South Africa in 1975, hepatocellular necrosis was the most pronounced pathologic finding (Figures 10, 11). The focal necrosis was similar to that in the Zaire Ebola cases, but the damage seemed almost synchronous, that is, large numbers of hepatocytes seemed to have been caught at the time of death of the patient in a similar state of early cytoplasmic hyalinization and eosinophilia without frank dissolution (Figures 12, 13). Small inclusions and Councilman like bodies were identified in this liver tissue (Figures 14, 15). There were necrotic cells in other organs, but not in numbers like those in the liver. In the kidney there was tubular necrosis and in glomerular capillaries there were multifocal fibrin thrombi characteristic of disseminated intravascular coagulopathy (DIC) 8 (Figure 16). There was also pulmonary edema and an effusion of macrophages into alveoli (Fi gure 17). It is hoped that a detailedpathologic description of this case will be published soon by South African pathologists.

Because study of the Marburg virus disease of 1967 was so comprehensive, the findings have special comparative value(1,2,3 ). Gross pathologic findings included evidences of hemorrhagic diatheses into skin, mucous membranes, soft tissues, visceral organs and into the stomach and intestines. There was swelling of spleen, lymph nodes, kidney and especially brain. Microscopically, focal necroses were found in many organs, most conspicuously in the liver, lymphatic system, testes, and ovaries. Liver necrosis, identical to that described in Ebola virus infection, was especially prominent, and as in the latter there was no favoring of anyparticular zone of lobules. Necrotic focigrew by expansion. Inclusion bodies were prominent, as were Councilmanlike bodies and basophilickaryorrhectic debris. Liver biopsies from convalescent patients indicated rapid regeneration coinciding with the decline of serum transaminase levels. Lymphoreticular organ changes included necrosis of 1) lymphoid follicles, 2) the red pulp of spleen, and 3) the medulla of lymph nodes. An eosinophilic "thrombic" debris was left in situ as a result of this necrosis. There were histologic evidences of hemorrhagic diathesis in many organs. In the brain there was a diffuse panencephalitis with glial nodule formation, perivascular lymphocytic cuffing, and evidence of interstitial edema. PathogenesisThe pathophysiologic alter ations which make Marburg and Ebola virus infections so devastating have not been studied systematically. The cause of the hemorrhagic diatheses was searched for intissues from the fatal Marburg cases in 1967 but no vascular lesions were identified. 

The I ncrease in vascular permeability, associated reduced effective circulating blood volume, interstitial edema in visceral organs and brain, and DIC may all stem from the liver necrosis and renal tubular necrosis. It is not clear how the shock syndrome in this disease relates to activities of pharmacologic mediators of capillary permeability and complement split products. Whatever the underlying mechanism, it was concluded after the 1967 Marburg episode that DIC and cerebral edema played an essential role in the  fatal outcome of infection. The magnitude and rapidity of the destructive events of Marburg or Ebola infection would predict that the terminal DIC/shock syndrome would be most difficult to deal with and  this is the case in fact. The success in South Africa in saving two Marburg patients with heparin and supportive treatment and the success in the United Kingdom in saving one Ebola patient with convalescent plasma does not tell us too much about the pathogenetic mechanisms of these hemorrhagic fevers, but they do remind us of the regenerative capacity of the liver and kidney tubules.

Differential Pathologic Diagnosis Although no pathognomonic lesion has been found which would permit certain histopathologic diagnosis of Marburg/Ebola infections, some pathologists considered that the overall pattern of lesions  is unique and distinguishable from 1,2 yellow fever, infectious hepatitis, and other well known infections. Others deem the differential diagnosis extremely difficult, especially in the context of examination of tissues from a single case and in a setting where Lassa Fever must also be considered. In any event, it is clear that pathologic examination is not a substitute for etiologic diagnosis(8), but inareas without full laboratory facilities, histolog ic diagnosis may be as important as it has been for many years in yellow fever diagnosis. The recent production by Drs. Y. Robin and J. Renaudet (with the support of the WHO) of an excellent 35mm transparency set with accompanying text, entitled 'La Fièvre Jaune histopathologique positif et differentiel', provides an important new resource for the differential pathologic diagnosis of hemorrhagic fevers in the African setting where Lassa fever, yellow .

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