2009 Pandemic Influenza A (H1N1)

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. 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. In April 2009, novel influenza A (H1N1) virus infection was first reported in two US children,1Dawood FS Jain S Finelli L Shaw MW Lindstrom S Garten RJ Gubareva LV Xu X Bridges CB Uyeki TM Emergence of a novel swine-origin influenza A (H1N1) virus in humans.N Engl J Med. 2009; 360: 2605-2615Crossref PubMed Scopus (2687) Google Scholar followed by identification of cases with acute respiratory illness caused by the identical virus infection in Mexico.2Echevarria-Zuno S Mejia-Arangure JM Mar-Obeso AJ Grajales-Muniz C Robles-Perez E Gonzalez-Leon M Ortega-Alvarez MC Gonzalez-Bonilla C Rascon-Pacheco RA Borja-Aburto VH Infection and death from influenza A H1N1 virus in Mexico: a retrospective analysis.Lancet. 2009; 374: 2072-2079Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar Global transmission of 2009 influenza A (H1N1) [2009 H1N1] virus led to the first influenza pandemic since 1968. 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We performed histopathological, immunohistochemical (IHC), and other laboratory methods to study viral distribution and cellular localization and to provide further insights into the pathogenesis of this disease. This study includes autopsy specimens from confirmed 2009 H1N1 case-patients submitted to the Infectious Diseases Pathology Branch, Centers for Disease Control and Prevention (CDC), for evaluation from May 1 to October 1, 2009. A confirmed case of 2009 H1N1 was defined as a patient with influenza-like illness and 2009 H1N1 virus infection confirmed by real-time RT-PCR (rRT-PCR) of either pre- or postmortem respiratory specimens at outside laboratories or CDC. Demographic data, laboratory test results for influenza virus and bacteria, and other relevant clinical information were collected from medical records and preliminary autopsy reports when available. Tissues were received as unfixed fresh or frozen, fixed in formalin, or as formalin-fixed, paraffin-embedded (FFPE) blocks. Autopsy specimens from 202 deaths were received by the Infectious Diseases Pathology Branch, and 104 of these were confirmed as described above. Four case-patients who did not have sufficient representative respiratory tissues available were excluded from the evaluation. Nonrespiratory tissues were evaluated from some case-patients, including heart (n = 30), liver (n = 23), brain (n = 19), kidney (n = 14), spleen (n = 12), gastrointestinal tract (n = 10), muscle (n = 4), and pancreas (n = 4). Nucleic acid extracts were prepared by using either fresh or frozen tissue samples or FFPE tissues. Briefly, for fresh or frozen tissues, a 1 to 3 mm3 portion of tissue was minced, and nucleic acids were extracted per protocol by using the RNeasy Mini Kit (Qiagen, Valencia, CA) for RNA and EZ1 DNA Tissue Mini Kit for DNA. For FFPE sections, the EZ1 RNA, DNA Tissue Mini Kits, MagAttract RNA, or DNA Tissue Mini M48 Kits (Qiagen) were used. The CDC rRT-PCR Protocol for Detection and Characterization of Swine Influenza, (http://www.who.int/csr/resources/publications/swineflu/realtimeptpcr/en/index.html, last accessed May 11, 2010), which universally detects influenza A viruses, swine influenza A viruses, and swine H1 influenza, was used to determine whether respiratory tissue samples from all case-patients and select nonrespiratory tissues were positive for 2009 H1N1. RNase P or β2-microglobulin gene targets were used as an internal control to ensure effective RNA extraction. In cases where the samples were identified as influenza A/unsubtypeable using this assay, primers and probes from a recently published assay that discriminates seasonal H1 from pandemic H1 in a single tube were used with modified cycling conditions.18Wang R Sheng ZM Taubenberger JK Detection of novel (swine origin) H1N1 influenza A virus by quantitative real-time reverse transcription-PCR.J Clin Microbiol. 2009; 47: 2675-2677Crossref PubMed Scopus (50) Google Scholar PCR assays for respiratory syncytial virus, parainfluenza viruses, and adenovirus were also performed by using the extracted nucleic acids. The DNA extracts from respiratory tissues from all case-patients were evaluated with a broad-range eubacterial PCR assay targeting the 16S rRNA gene.19Imrit K Goldfischer M Wang J Green J Levine J Lombardo J Hong T Identification of bacteria in formalin-fixed, paraffin-embedded heart valve tissue via 16S rRNA gene nucleotide sequencing.J Clin Microbiol. 2006; 44: 2609-2611Crossref PubMed Scopus (40) Google Scholar Conventional single stage or nested PCR assays for Streptococcus pneumoniae targeting the pneumolysin gene20Murdoch DR Anderson TP Beynon KA Chua A Fleming AM Laing RT Town GI Mills GD Chambers ST Jennings LC Evaluation of a PCR assay for detection of Streptococcus pneumoniae in respiratory and nonrespiratory samples from adults with community-acquired pneumonia.J Clin Microbiol. 2003; 41: 63-66Crossref PubMed Scopus (87) Google Scholar; Streptococcus pyogenes targeting the Streptococcal pyrogenic exotoxin B (speB) gene21Guarner J Sumner J Paddock CD Shieh WJ Greer PW Reagan S Fischer M Van Beneden CA Zaki SR Diagnosis of invasive group a streptococcal infections by using immunohistochemical and molecular assays.Am J Clin Pathol. 2006; 126: 148-155Crossref PubMed Scopus (15) Google Scholar; and Haemophilus influenzae targeting the outer membrane protein P6 gene22Stralin K Backman A Holmberg H Fredlund H Olcen P Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, and Chlamydophila pneumoniae to be used on sputum samples.APMIS. 2005; 113: 99-111Crossref PubMed Scopus (67) Google Scholar were performed by using DNA from select case-patients. Amplified PCR products were sequenced on a CEQ 8000 automated sequencer (Beckman Coulter, Fullerton, CA) for confirmation. DNA extracts from select cases were tested for methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) with two real-time multiplex PCR assays: (1) S. aureus toxin genes PCR assay targets Panton-Valentine Leukocidin gene and toxic shock syndrome toxin 1 gene; (2) PCR assay, which detects mecA (exogenous gene encoding methicillin resistance in staphylococci), nuc (heat stable DNA nuclease, specific for S. aureus), and femA (factor essential for methicillin resistance, specific for Staphylococcus epidermidis) genes.23Thomas LC Gidding HF Ginn AN Olma T Iredell J Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture.J Microbiol Methods. 2007; 68: 296-302Crossref PubMed Scopus (83) Google Scholar The presence of amplifiable DNA in all extracts was verified by amplification of human house-keeping genes β-globin and glyceraldehyde-3-phosphate dehydrogenase. Routine H&E stains of available major organs were evaluated for histopathological changes. Lillie-Twort Gram stain and Warthin-Starry silver impregnation stain were performed on selected sections of all case-patients to examine for the presence of bacterial infection. IHC assays were performed by using a polymer-based colorimetric indirect immunoalkaline phosphatase method. Respiratory and available nonrespiratory tissues from all case-patients were evaluated with a monoclonal antibody against the nucleoprotein of influenza A virus.24Guarner J Shieh WJ Dawson J Subbarao K Shaw M Ferebee T Morken T Nolte KB Freifeld A Cox N Zaki SR Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs.Am J Clin Pathol. 2000; 114: 227-233Crossref PubMed Scopus (77) Google Scholar Select respiratory sections from all case-patients were evaluated with a polyclonal antibody against Group A Streptococcus (S. pyogenes)21Guarner J Sumner J Paddock CD Shieh WJ Greer PW Reagan S Fischer M Van Beneden CA Zaki SR Diagnosis of invasive group a streptococcal infections by using immunohistochemical and molecular assays.Am J Clin Pathol. 2006; 126: 148-155Crossref PubMed Scopus (15) Google Scholar; a polyclonal antibody against S. pneumoniae25Guarner J Packard MM Nolte KB Paddock CD Shieh WJ Tondella ML McGee L Zaki SR Usefulness of immunohistochemical diagnosis of Streptococcus pneumoniae in formalin-fixed, paraffin-embedded specimens compared with culture and gram stain techniques.Am J Clin Pathol. 2007; 127: 612-618Crossref PubMed Scopus (19) Google Scholar; a polyclonal antibody against H. influenzae; and a monoclonal antibody against S. aureus. The antibody/polymer conjugate was visualized by applying UltraVision LP system with Napthol Phosphate Substrate (Thermo Scientific/Lab Vision) to tissue sections. Negative controls consisted of sequential tissue sections incubated with normal serum pertinent to the primary antibody. Double-stain IHC was performed by using peroxidase polymer-labeled antibodies (Dako, Carpinteria, CA) against cytokeratin, surfactant, or CD68, followed by the mouse anti-influenza A nucleoprotein antibody labeled with immunoalkaline phosphatase polymer.26Shieh WJ Hsiao CH Paddock CD Guarner J Goldsmith CS Tatti K Packard M Mueller L Wu MZ Rollin P Su IJ Zaki SR Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan.Hum Pathol. 2005; 36: 303-309Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Formalin fixed tissues or FFPE sections from five cases with duration of illness <7 days and abundant immunostaining were processed for transmission electron microscopy as previously described.27Estrada JC Selim MA Miller SR TEM of tissue from paraffin-embedded H&E-stained 6-micron sections for diagnosis (an unusual papovavirus case).Microsc Microanal. 2005; 11: 964CD-965CDGoogle Scholar, 28Goldsmith CS Whistler T Rollin PE Ksiazek TG Rota PA Bellini WJ Daszak P Wong KT Shieh WJ Zaki SR Elucidation of Nipah virus morphogenesis and replication using ultrastructural and molecular approaches.Virus Res. 2003; 92: 89-98Crossref PubMed Scopus (61) Google Scholar Confluent monolayers of MDCK cells grown in Dulbecco’s modified Eagle’s medium were inoculated with fresh respiratory tissue and monitored daily for cytopathic effect. Immunofluorescence testing by using the monoclonal antibody against nucleoprotein was performed when cytopathic effect was identified or 8 days postinoculation if no cytopathic effect was observed. One hundred confirmed case-patients with fatal 2009 H1N1 virus infection were evaluated at the Infectious Diseases Pathology Branch during May 12 to October 1, 2009. Fifty-three (53%) of these case-patients had 2009 H1N1 confirmed by postmortem specimen testing. The median age of fatal case-patients was 36 years (range, 2 months to 84 years), 80% were aged 20 to 60 years, and 51 (51%) were male patients (Table 1). The majority (85%) of case-patients with known previous medical history had at least one underlying comorbidity. Obesity (46%), cardiovascular disease (25%), and asthma (22%) were the three most frequent conditions reported. Fever (82%), cough (67%), and shortness of breath (58%) were the most common signs and symptoms reported, with a median duration from illness onset to death of 8 days (range, 1 to 44 days). Fifty-eight (67%) of 87 case-patients with available clinical history were hospitalized before death. Forty-two (74%) of 57 case-patients with available hospital records required mechanical ventilation. Radiographical diagnosis of pneumonia was documented in 59% (38 of 64) of case-patients.Table 1Characteristics of Confirmed 2009 H1N1 Case-Patients with Autopsy Samples Evaluated for Pathologic FeaturesCharacteristicNo. (%)Male patients51 (51)Age group 0–94 (4) 10–1913 (13) 20–2917 (17) 30–3924 (24) 40–4922 (22) 50–5917 (17) ≥603 (3)Medical history (n = 93) At least one pre-existing condition79 (85) Obesity*Obesity is defined as body mass index ≥30 and extreme obesity as body mass index ≥40.43 (46) Extreme obesity*Obesity is defined as body mass index ≥30 and extreme obesity as body mass index ≥40.16 (17) Cardiovascular disease23 (25) Asthma20 (22) Diabetes12 (13) Pregnancy6 (6) HIV infection4 (4)Duration of illness, median days (range)8 (1–44 days)Clinical symptoms (n = 72) Fever59 (82) Cough48 (67) Shortness of breath42 (58) Headache11 (15) Fatigue/weakness10 (14) Sore throat11 (15) Vomiting16 (22) Diarrhea8 (11)Hospitalized (n = 87)58 (67)Invasive mechanical ventilation (n = 57)42 (74)Antiviral treatment (n = 67)44 (66)* Obesity is defined as body mass index ≥30 and extreme obesity as body mass index ≥40. Open table in a new tab Table 2 presents the histopathological features observed in airways and lungs. Eighty-five case-patients had airway tissues available for evaluation. The most frequent histopathological findings in airways were inflammation and edema (66%). The inflammation was usually mild and consisted predominantly of mononuclear cells (Figure 1A). Necrosis of epithelium (26%) and hemorrhage (18%) were less frequently observed. Lung tissues in all case-patients showed a spectrum of histopathological changes of diffuse alveolar damage (DAD), including edema, hyaline membranes, inflammation, and fibrosis (Figure 1, B–G). The nature and extent of DAD generally corresponded to the duration of clinical illness of the patients. Forty-one case-patients had paratracheal or hilar lymph nodes available for evaluation, and hemophagocytosis was noted in 25 (61%) of these case-patients. Pulmonary thromboemboli were noted in gross autopsy findings or microscopically in 17 case-patients. No histopathological evidence of myocarditis or encephalitis was observed in any of the case-patients with heart (n = 30) or brain samples (n = 19) available for evaluation. Histopathological findings in other organs were nonspecific and likely associated with the patients’ underlying medical conditions. These findings included prominent eosinophils in patients with history of asthma, enlarged nuclei of cardiac myocytes in patients with history of hypertension, and fatty metamorphosis in the liver in obese patients.Table 2Histopathological Features in Respiratory Tract Specimens of 2009 H1N1 Fatal Case-Patients (N = 100)Histopathologic featureNo. (%) of case-patients with featureTrachea and bronchi (n = 85) Inflammation and edema56 (66) Necrosis22 (26) Hemorrhage15 (18)Lung*Edema, fibrin, and hemorrhage were seen in alveolar spaces; inflammation and organizing fibrosis primarily involved interstitium but were also focally seen within alveolar spaces. (n = 100) Edema63 (63) Hyaline membranes59 (59) Fibrin58 (58) Hemorrhage58 (58) Inflammation48 (48) Type II pneumocyte hyperplasia46 (46) Neutrophilic bronchopneumonia29 (29) Organizing fibrosis28 (28) Squamous metaplasia12 (12)* Edema, fibrin, and hemorrhage were seen in alveolar spaces; inflammation and organizing fibrosis primarily involved interstitium but were also focally seen within alveolar spaces. Open table in a new tab The IHC assay results for influenza A were positive in respiratory tissues from 44 of the 100 case-patients (44%); the amount of influenza virus antigen varied, with abundant immunostaining in nine case-patients and rare in 24 case-patients. Viral nucleoprotein antigens were localized in the nuclei and cytoplasm of infected cells, including epithelial cells in airways (Figure 2A), submucosal glands (Figure 2B), and pneumocytes, either detached or lining alveoli (Figure 2, C–E). Antigens were also seen in association with hyaline membranes (Figure 2F) and in endothelial cells in rare cases (Figure 2G). Double staining revealed that the major cellular targets of viral infection were pneumocytes (co-labeled with cytokeratin; Figure 2H), predominantly type II (co-labeled with surfactant; Figure 2I), and occasionally macrophages (co-labeled with CD68; Figure 2J). No immunostaining of influenza A viral antigen was detected in any of the nonrespiratory tissue samples available. Influenza rRT-PCR testing on a limited number of these nonrespiratory samples was also negative. Electron microscopic examination of lung tissue identified rare infected cells with extracellular virus particles in the alveolar space (Figure 2K). Virions were round to oblong-shaped and averaged 88 nm in diameter; some particles were surrounded by spikes approximately 12 nm in length (Figure 2L). Overall, 26 (26%) case-patients had confirmatory test results of bacterial co-infection. Twenty-nine case-patients showed histopathological evidence of bronchopneumonia with prominent alveolar polymorphonuclear cells, indicating a possibility of bacterial co-infection (Figure 3A, D, and G). Of these, 22 (76%) case-patients had a specific bacterial pathogen identified. Bacterial agents were identified in an additional four case-patients that did not show histopathological evidence of bronchopneumonia in the tissue sections examined. Gram-positive cocci were the most frequent bacteria identified by using special stains (Figure 3C and F). IHC and PCR assays were positive as follows: nine case-patients positive for S. pneumoniae (Figure 3B); three case-patients for S. pyogenes (Figure 3E); one case-patient for both S. pyogenes and S. pneumoniae; one case-patient for both S. pyogenes and S. mitis; one case-patient for S. mitis; one case-patient for S. agalactiae; four case-patients for MRSA (Figure 3H); one case-patient for both MRSA and S. pyogenes; one case-patient for both MRSA and H. influenzae; and four case-patients for MSSA. None of the case-patients were found to have evidence of a co-infection with respiratory syncytial virus, parainfluenza viruses 1 to 3, or adenovirus. Of the 100 case-patients with confirmed 2009 H1N1, testing of respiratory tissue by rRT-PCR assays at CDC were positive for influenza A virus in 90, including 87 for H1N1 and 3 nonsubtypeable virus (Table 3). Based on available records for 80 case-patients with known duration of illness, rRT-PCR results were positive for 2009 H1N1 in respiratory tissue specimens of 42 (53%) case-patients with illness duration <10 days and in 28 case-patients (35%) with illness >10 days when death occurred. Negative rRT-PCR results for 2009 H1N1 were not observed in any case-patients with known illness duration <10 days, but results were negative in seven case-patients (9%) with duration >10 days. In these same 80 case-patients with known duration of illness, positive IHC results for influenza A viral antigen were observed in respiratory tissues of 31 case-patients (39%) with illness duration <10 days and in five case-patients (6%) with illness >10 days. In contrast, negative IHC results for influenza A were observed in respiratory tissues of 14 case-patients (18%) with time from onset to death <10 days and in 31 case-patients (39%) who died after an illness >10 days. The 2009 H1N1 virus was isolated from fresh lung tissue specimens in 5 of 30 case-patients tested.Table 3Diagnostic Results for Respiratory Tissues Evaluated from 100 Fatal Cases of 2009 H1N1Influenza ABacterial co-infectionsTotal no. of case-patientsRT-PCR*Of the 100 case-patients included in this study, 90 were positive for influenza A in respiratory tissue evaluated, either for 2009 H1N1 (+) or nonsubtypeable (A/unsub). The 10 case-patients that were PCR negative (−) were confirmed by outside laboratory results from clinical samples.IHCPCRIHC+−−−33++−−29++++14+−++10++−+1A/unsub−++2A/unsub−−−1−−−−8−−++1−−−+1Total100* Of the 100 case-patients included in this study, 90 were positive for influenza A in respiratory tissue evaluated, either for 2009 H1N1 (+) or nonsubtypeable (A/unsub). The 10 case-patients that were PCR negative (−) were confirmed by outside laboratory results from clinical samples. Open table in a new tab This report presents pathological studies on autopsy samples from 100 patients with fatal 2009 H1N1 virus infection that occurred between May and October 2009 in the United States. Histopathological evaluations revealed DAD as the most significant and consistent finding, and immunolocalization showed viral antigens predominantly in the lung parenchyma. This is somewhat similar to the pattern observed in fatal case-patients of highly pathogenic avian influenza (H5N1).29Gu J Xie Z Gao Z Liu J Korteweg C Ye J Lau LT Lu J Zhang B McNutt MA Lu M Anderson VM Gong E Yu AC Lipkin WI H5N1 infection of the respiratory tract and beyond: a molecular pathology study.Lancet. 2007; 370: 1137-1145Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 30Korteweg C Gu J Pathology, molecular biology, and pathogenesis of avian influenza A (H5N1) infection in humans.Am J Pathol. 2008; 172: 1155-1170Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 31Ng WF To KF Pathology of human H5N1 infection: new findings.Lancet. 2007; 370: 1106-1108Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 32To KF Chan PK Chan KF Lee WK Lam WY Wong KF Tang NL Tsang DN Sung RY Buckley TA Tam JS Cheng AF Pathology of fatal human infection associated with avian influenza A H5N1 virus.J Med Virol. 2001; 63: 242-246Crossref PubMed Scopus (385) Google Scholar However, a significant proportion of 2009 H1N1 case-patients in this report also showed viral localization along with inflammation or other histopathological changes in trachea, bronchi, or bronchioles (Table 2), a pattern more commonly seen in severe or fatal cases of seasonal influenza.11Guarner J Paddock CD Shieh WJ Packard MM Patel M Montague JL Uyeki TM Bhat N Balish A Lindstrom S Klimo