Pathological and ultrastructural analysis of surgical lung biopsies in patients with swine-origin influenza type A/H1N1 and acute respiratory failure

Abstract

Background: Cases of H1N1 and other pulmonary infections evolve to acute respiratory failure and death when co-infections or lung injury predominate over the immune response, thus requiring early diagnosis to improve treatment.

Objective: To perform a detailed histopathological analysis of the open lung biopsy specimens from five patients with ARDS with confirmed H1N1.

Methods: Lung specimens underwent microbiologic analysis, and examination by optical and electron microscopy. Immunophenotyping was used to characterize macrophages, natural killer, T and B cells, and expression of cytokines and iNOS.

Results: The pathological features observed were necrotizing bronchiolitis, diffuse alveolar damage, alveolar hemorrhage and abnormal immune response. Ultrastructural analysis showed viral-like particles in all cases.

Conclusions: Viral-like particles can be successfully demonstrated in lung tissue by ultrastructural examination, without confirmation of the virus by RT-PCR on nasopharyngeal aspirates. Bronchioles and epithelium, rather than endothelium, are probably the primary target of infection, and diffuse alveolar damage the consequence of the effect of airways obliteration and dysfunction on innate immunity, suggesting that treatment should be focused on epithelial repair.

Lung sections from N1H1 patients examined by Hematoxilyn Eosin staining. This panel show pulmonary parenchyma modified by extensive alveolar lesion characterized by alveolar hemorrhage (A), disproportionately sparse infiltrate of inflammatory cells (B) and collapse alveolar areas with dilatation of the airspaces (C). In other areas show diffuse alveolar damage with interstitial thickening (D), mild to moderate fibroplasia (E), hyaline membrane formation (F) and intra‐alveolar fibrin exudates (G to I). Intra‐alveolar fibrin exudates similar with moderate infiltrate of inflammatory cells characterized by histiocytes, lymphocytes, megakariocytes and multinucleated forms (J). These atypical forms included multinucleated giant cells with granular amphophilic cytoplasm, irregular, large and atypical nuclei and prominent eosinophilic nucleoli (arrows) (K to M), (Hematoxilyn Eosin).

Ultrastructure sections of lungs from N1H1 patients. Large number of bronchial (EC) and alveolar epithelial cells (AEC2) in apoptosis, note markedly condensed chromatin close to the nuclear membrane (A to C). In panel D, denudation of the epithelial basement membrane and fibrin deposition (arrows). In alveoli, the hyaline membranes appear homogeneous and extend along and partially cover the denuded epithelial basement membranes (D). Regeneration of epithelial cells is shown in panel E to I: note the cells with prominent surface microvilli with decreased or absent lamellar bodies and considerable cytologic atypia. Myofibroblasts (MY) and collagen fibers (COL) were also present (E,F,G). Multinucleated epithelial cells (J,K,L) showing tubuloreticular structures (TRS) and cylindrical confronting cisternae (CCC) representing viral‐like particles residuals are shown in M (arrow head), N (star), O (arrow head). TRS showing reticular aggregates of branching membranous tubules located within the cisternae of the endoplasmic reticulum (P,Q,R). Cylindrical confronting cisternae (CCC) showing long cylinders of fused membranous lamellae (M to R), (Uranile and lead citrate).

Immunologic features of lungs from N1H1 patients. Small aggregates of CD68 (A), CD4+ (B), CD8+ (C), CD20+ (D), NK+ (E), and S100+ (F) present around vessels and bronchioles. Note a very strong expression of IL‐4 (G), IL‐10 (H) and iNOS (I) in macrophages and small mononucleated cells, (Immmunohistochemistry).