2009 pandemic influenza A (H1N1): pathology and pathogenesis of 100 fatal cases in the United States

Abstract

In the spring of 2009, a novel influenza A (H1N1) virus emerged in North America and spread worldwide to cause the first influenza pandemic since 1968. During the first 4 months, over 500 deaths in the United States had been associated with confirmed 2009 pandemic influenza A (H1N1) [2009 H1N1] virus infection. Pathological evaluation of respiratory specimens from initial influenza-associated deaths suggested marked differences in viral tropism and tissue damage compared with seasonal influenza and prompted further investigation. Available autopsy tissue samples were obtained from 100 US deaths with laboratory-confirmed 2009 H1N1 virus infection. Demographic and clinical data of these case-patients were collected, and the tissues were evaluated by multiple laboratory methods, including histopathological evaluation, special stains, molecular and immunohistochemical assays, viral culture, and electron microscopy. The most prominent histopathological feature observed was diffuse alveolar damage in the lung in all case-patients examined. Alveolar lining cells, including type I and type II pneumocytes, were the primary infected cells. Bacterial co-infections were identified in >25% of the case-patients. Viral pneumonia and immunolocalization of viral antigen in association with diffuse alveolar damage are prominent features of infection with 2009 pandemic influenza A (H1N1) virus. Underlying medical conditions and bacterial co-infections contributed to the fatal outcome of this infection. More studies are needed to understand the multifactorial pathogenesis of this infection.

Figure 1

Histopathological findings in airway and lung; H&E staining. Original magnification: ×50 (A, B, E, and G); ×25 (C and D); ×100 (F). A: Inflammation in the airway is usually mild and predominantly composed of mononuclear cells. B: The lungs show various phases of diffuse alveolar damage, including fibrinous inflammation, intra-alveolar hemorrhage (C), intra-alveolar edema and hyaline membrane formation (D and E), type II pneumocytes hyperplasia (F), and organizing fibrosis (G).

Figure 2

Immunolocalization of 2009 H1N1 influenza viral antigens and ultrastructural features: immunoalkaline phosphate staining, naphthol fast red substrate with light hematoxylin counterstain (A–G); double immunostaining with immunoperoxidase and immunoalkaline phosphatase, naphthol fast red substrate with light hematoxylin counterstain (H–J); and thin-sectioned electron microscopy (K and L). Original magnification: ×50 (A, D, and F); ×25 (B); ×12.5 (C); ×100 (E and G); ×158 (H–J). Scale bars = 500 nm (K); 100 nm (L). A: Viral antigens are detected in the nuclei of infected airway epithelial cells. B: Viral antigens are seen in submucosal glands. C–E: Viral antigens are predominantly present in the nuclei of alveolar lining cells, including type I and type II pneumocytes; many of them have detached and are in the alveolar space. F: Immunostaining of viral antigens is seen in hyaline membranes lining alveoli. G: Viral antigens are located in endothelial cells. H and I: Double staining reveals influenza viral antigens (brown) are predominantly in pneumocytes (red, co-labeled with cytokeratin [H]), specifically in type II pneumocytes (red, co-labeled with surfactant [I]). Viral antigen (brown) can also be seen in occasional macrophages (red, co-labeled with CD68 [J]). K: Electron micrograph showing infected cell (asterisk) with extracellular virus particles, associated with dense material. L: Virions consist of internal nucleocapsids surrounded by an envelope with surface projections.

Figure 3

Bacterial co-infections detected with histopathology, special stains, and IHC. A, D, and G: H&E staining; B, E, and H: immunoalkaline phosphate staining, naphthol fast red substrate with light hematoxylin counterstain; C and F: Lilly-Twort tissue Gram staining. Original magnification: ×25 (A, D, and G); ×100 (B and E); ×158 (C and F); ×50 (H). A: Abundant neutrophilic infiltrate in the alveoli indicative of an acute pneumonic process. B: Extracellular and intracellular immunostaining of S. pneumoniae antigens. C: Gram-positive cocci in the serial section as B. D: Abundant neutrophilic infiltrate in the alveoli of another patient with pneumonia. E: Extracellular and intracellular immunostaining of Group A Streptococcus antigens. F: Gram-positive cocci in the serial section as E. G: Prominent necrosis and neutrophilic infiltrate indicative of a necrotizing pneumonia. H: Abundant immunostaining of S. aureus.