Lung Pathology in Fatal Novel Human
Influenza A (H1N1) Infection
Thais Mauad1, Ludhmila A. Hajjar2, Giovanna D. Callegari1, Luiz F. F. da Silva1, Denise Schout3,
Filomena R. B. G. Galas2, Venancio A. F. Alves1, Denise M. A. C. Malheiros1, Jose O. C. Auler, Jr.2,
Aurea F. Ferreira1, Marcela R. L. Borsato1, Stephania M. Bezerra1, Paulo S. Gutierrez4,
Elia T. E. G. Caldini1, Carlos A. Pasqualucci1,5, Marisa Dolhnikoff1, and Paulo H. N. Saldiva1
1Department of Pathology,2Department of Anesthesiology,3Department of Preventive Medicine, Epidemiology Service, Hospital das Clı ´nicas,
4Laboratory of Pathology—Heart Institute, Hospital das Clı ´nicas, and5Autopsy Service of Sao Paulo City, Sa ˜o Paulo University, Sa ˜o Paulo, Brazil
Rationale: There are no reports of the systemic human pathology of
the novel swine H1N1 influenza (S-OIV) infection.
Objectives: The autopsy findings of 21 Brazilian patients with con-
firmed S-OIV infection are presented. These patients died in the
winter of the southern hemisphere 2009 pandemic, with acute
Methods: Lung tissue was submitted to virologic and bacteriologic
analysis with real-time reverse transcriptase polymerase chain
reaction and electron microscopy. Expression of toll-like re-
ceptor (TLR)-3, IFN-g, tumor necrosis factor-a, CD81T cells and
granzyme B1cells in the lungs was investigated by immunohisto-
in 20 individuals. In six patients, diffuse alveolar damage was
associated with necrotizing bronchiolitis and in five with extensive
alveolar epithelial cells, as well as necrosis, epithelial hyperplasia,
and squamous metaplasia of the large airways. There was marked
granzyme B1cells within the lung tissue. Changes in other organs
were mainly secondary to multiple organ failure.
Conclusions: Autopsies have shown that the main pathological
changes associated with S-OIV infection are localized to the lungs,
where three distinct histological patterns can be identified. We
also show evidence of ongoing pulmonary aberrant immune
response. Our results reinforce the usefulness of autopsy in increas-
ing the understanding of the novel human influenza A (H1N1)
Keywords: autopsy; virus; diffuse alveolar damage; bronchiolitis; innate
In April 2009, a novel swine-origin influenza A (H1N1) virus (S-
OIV) was identified in California and Mexico as a cause of
human respiratory disease (1–3). In June 2009, the World
Health Organization signaled that a novel H1N1 flu pandemic
was underway (4). As of October 11, 2009, more than 399,232
confirmed cases of novel H1N1 influenza virus and at least 4,735
deaths have been reported globally (5, 6).
The pandemic novel influenza A (H1N1) infection was
considered widespread in Brazil on July 16. As of October 10,
2009, there were 17,219 cases confirmed in Brazil, including
1,368 deaths, most of them concentrated in Sao Paulo state (7).
The Hospital das Clinicas of the University of Sao Paulo is the
largest tertiary health care center in Brazil and a reference
center for H1N1 cases in Sa ˜o Paulo. From June through
October 2009, there were 494 confirmed cases of S-OIV in-
fection in this hospital, of which 223 were admitted due to
respiratory symptoms and 16 died.
Most patients with S-OIV infection present flulike symptoms
with a benign course. However, patients with comorbidities may
have a serious clinical presentation with respiratory failure (1,
8–10). The main cause of death is acute respiratory distress
syndrome (ARDS) (1).
Previous studies on experimental influenza infections have
shown that the pattern-recognition toll-like receptor (TLR) 3
initiates a proinflammatory response of lung epithelial cells to
influenza virus–derived dsRNA (11). CD81T cells are impor-
tant in the antiviral response to influenza via direct lysis or by
the induction of tumor necrosis factor (TNF)-a and IFN-g.
Altered innate immune responses, excess of CD81T-cell
cytotoxic responses, and hypercytokinemia are related to dis-
ease severity in influenza infections (12, 13).
There are no existing reports on the systemic pathology of
patients who died with the S-OIV infection. The lack of
information on the pathophysiology of this novel disease is
a limitation that prevents better clinical management and
hinders the development of a therapeutic strategy. We hypoth-
esize that a deregulation in antiviral immune response pathways
could occur in the lungs and contribute to severity of the
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Most patients with H1N1 infection present flulike symp-
toms with a benign course. Patients with comorbidities may
have a serious clinical presentation with respiratory failure.
The main cause of death is acute respiratory distress
syndrome. The pulmonary pathology of patients who died
with the H1N1 infection is incompletely described.
What This Study Adds to the Field
This study shows that the main pathological changes asso-
ciated with H1N1 infection are localized to the lungs, where
three distinct histological patterns can be identified: diffuse
alveolar damage (DAD), necrotizing bronchiolitis, and
DAD with intense alveolar hemorrhage. There is also evi-
dence of ongoing pulmonary aberrant immune response.
(Received in original form September 22, 2009; accepted in final form October 27, 2009)
Supported by the Conselho Nacional de Desenvolvimento Cientı ´fico e Techno-
lo ´gico (CNPq), Brazil.
Correspondence and requests for reprints should be addressed to Thais Mauad
M.D., Ph.D., Department of Pathology, Sa ˜o Paulo University Medical School, Av.
Dr. Arnaldo, 455 room 1155, CEP 01246-903 Sa ˜o Paulo SP, Brazil. E-mail:
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.200909-1420OC on October 29, 2009
Internet address: www.atsjournals.org
Vol 181. pp 72–79, 2010
Hence, it is timely to report pathological findings of S-OIV
infection. Here, we describe autopsy findings, with an emphasis
on lung immunopathology, of 21 Brazilian patients who died of
respiratory failure related to S-OIV infection.
This report was approved by the institutional Medical Ethical Com-
Twenty-one patients who died with a confirmed S-OIV infection had
their autopsies performed in the Department of Pathology of the
Hospital das Clinicas of the University of Sao Paulo during July and
August of 2009. This department is associated with the Autopsy
Service of Sao Paulo city, the largest service of this kind in Latin
America, where 13,000 autopsies are performed yearly (14). Of the
21 patients, 9 died in Hospital das Clinicas and 12 in other hospitals in
Clinical features were obtained from medical charts and from the
National Epidemiologic Surveillance System of the Ministry of Health.
Information was gathered regarding demographic data, preexisting
medical conditions, presentation of the viral disease, and evolution
The in vivo diagnosis of S-OIV infection was confirmed in
nasopharyngeal swab specimens by using the real-time reverse tran-
scriptase polymerase chain reaction (rRT-PCR) test, in accordance
with guidelines from the Centers for Disease Control and Prevention
Tissue fragments were formalin-fixed, paraffin-embedded, and hema-
toxylin and eosin–stained. For lung sections, Grocott, Brown-Hopps,
and Ziehl-Neelsen stainings were performed for the identification of
fungi, bacteria, and acid-fast bacilli, respectively.
Lung fragments were sent for microbiological investigation to the
Instituto Adolfo Lutz in Sao Paulo using rRT-PCR. Seasonal influenza
A and swine influenza A detection were performed using the CDC
protocol (15). The RT-PCR test used for bacteria identified DNA from
Haemophilus influenzae and Streptococcus pneumoniae.
Immunohistochemistry and Electron Microscopy
Immunostaining (16) for the following markers was analyzed in the
lung tissue of 13 patients infected with S-OIV and in 4 histologically
normal control lungs obtained from nonsmoking individuals who died
of nonpulmonary and noninfectious causes (16): TLR-3, TNF-a, IFN-g
(Santa Cruz Biotechnology, Santa Cruz, CA), CD81T cells (Dako,
Carpinteria, CA) and granzyme B1cells (Novocastra, Newcastle, UK).
In one patient who presented multiple giant cells in the lungs, we
performed immunohistochemistry for respiratory syncytial virus
(Novocastra) and herpes I and II (DAKO, Glostrup, Denmark).
Anti-myoglobin (DAKO) immunostaining was performed on kidney
Pulmonary tissue from three cases was fixed for electron micros-
copy as previously described (17).
Table 1 shows the clinical and epidemiological characteristics of
the 21 patients. Patient ages ranged from 1 to 68 years (median,
34 yr). Fifteen patients (72%) were between 30 and 59 years
old. Twelve patients (57%) were male. All patients resided in
Sao Paulo city. Sixteen patients (76%) had preexisting medical
conditions. Seven patients (33%) had at least one of the
following chronic cardiovascular diseases: systemic arterial
hypertension (5), left ventricular hypertrophy (4), chronic heart
failure (2), chronic coronary disease (1), and congenital heart
disease (1). Five patients (24%) had one of the following
cancers: myelofibrosis, esophageal, bowel, breast, or laryngeal
cancer. Six (29%) were current smokers. Two patients were
children; one was a 2-year-old boy with cyanotic complex
congenital heart disease, and the other was a 1-year-old boy
without previous disease. Five patients (24%), including a pre-
viously healthy pregnant woman at 28 weeks of gestational age,
did not have any identified comorbidity.
Most patients presented with dyspnea (86%), fever (71%),
myalgia (67%), and cough (57%). All patients had respiratory
failure requiring mechanical ventilation. Sixteen (76%) were
admitted to an intensive care unit; the other five died in
emergency services. In 15 patients (71%), the diagnosis of
ARDS was established based on the presence of bilateral
pulmonary infiltrates, PO2/FIO2less than or equal to 200, and
no clinical evidence for an elevated left atrial pressure (18).
In the other six patients who needed mechanical ventilation,
patients had acute lung injury but ARDS was not fully
TABLE 1. CHARACTERISTICS OF 21 PATIENTS WHO DIED OF
CONFIRMED INFECTION WITH NOVEL SWINE-ORIGIN
INFLUENZA A (H1N1) VIRUS
Chronic cardiovascular disease
Chronic respiratory disease
Chronic renal disease
Bone marrow transplantation
Respiratory failure requiring mechanical ventilation
Admission in intensive care unit
Acute respiratory distress syndrome
Acute renal failure
Acute renal failure with dialysis
Shock needing vasopressors
Treatment with oseltamivir
Treatment with antibiotic
Median of days from admission to death (range)
Clinically suspected bacterial pneumonia
rRT-PCR in nasopharyngeal swab for S-OIV
Bacteria isolation in culture*
Definition of abbreviations: rRT-PCR 5 real time reverse transcriptase poly-
merase chain reaction.
* Bacteria culture was obtained in bronchial aspirate samples in 9 patients.
Mauad, Hajjar, Callegari, et al.: Lung Pathology in H1N1 Infection 73
characterized. In nine patients for whom information was
available, the median Sequential Organ Failure Assessment
score was 7 (range, 3–14). Nine patients developed acute renal
failure and four required dialysis.
Sixteen patients (76%) received treatment with oseltamivir.
The median period from admission to death was 6 days (range,
1–12 d). Corticosteroid therapy was prescribed in 12 (57%)
patients. Sixty-two percent of patients received empiric treat-
ment for bacterial infection. Bacterial infection was confirmed
in bronchial aspirate samples in three out of nine patients; all
three had S. pneumoniae (# 4, 7, and 15, Table 2). S-OIV
infection was confirmed in all 21 patients by rRT-PCR testing of
Macroscopic examination revealed heavy and consolidated
lungs that were diffusely edematous with variable degrees of
hemorrhage (Figure 1).
TABLE 2. LUNG HISTOPATHOLOGICAL DATA OF 21 PATIENTS WHO DIED OF CONFIRMED INFECTION WITH NOVEL SWINE-ORIGIN
INFLUENZA A (H1N1) VIRUS
EffectPerivasculitis MicrothrombiPTE Comorbidity
Necrotizing bronchiolitis with exudative DAD
1 Moderate Moderate
2 Moderate Moderate
——— Present None
ModerateMild MildIntenseIntense— Present—— Culture of
MildModerateModerate ModerateModerate— Present—— SAH, ventricular
ModerateModerateModerateMild Moderate————Tissue PCR
Exudative DAD with intense alveolar hemorrhage
7 MildIntenseIntenseMild Moderate—— Present— Myelofibrosis,
11 MildMild IntenseMildModerate—Present——
12MildMild MildMildMild————SAH, ventricular
SAH, heart failure,
15—Intense —MildMild———— Histochemistry
No virus-related pulmonary changes
21———MildIntense——PresentPresent Laryngeal cancer—
Definition of abbreviations: DAD 5 diffuse alveolar damage; PTE 5 pulmonary thromboembolism; SAH 5 systemic arterial hypertension; PCR 5 polymerase chain
Em dashes (—) indicate absent.
* Evidence of bacterial infection was considered when patient presented positive histochemical staining for bacteria and/or positive culture in bronchial aspirate and/or
positive PCR. Both PCR and bronchial aspirate cultures detected Streptococcus pneumoniae in six patients.
74AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1812010
The details of the pathological findings in the lungs are
shown in Table 2 and Figure 1. All but one subject had
exudative diffuse alveolar damage (DAD). There were, how-
ever, three distinct patterns of pulmonary pathological changes
identified: (1) Nine patients had classic exudative DAD, with
alveolar and interstitial edema, alveolar fibrinous exudate with
hyaline membranes, and reactive pneumocytes. In these pa-
tients, interstitial inflammation was not prominent. (2) Six had
severe necrotizing bronchiolitis (NB) characterized by extensive
necrosis of the bronchiolar wall and dense neutrophilic infiltrate
within the bronchiolar lumen. In these patients, the DAD
showed a significant degree of exudate organization. Parenchy-
mal inflammation in these patients was severe and predomi-
nantly neutrophilic. (3) Five patients presented with exudative
DAD with an intense hemorrhagic component. In group three,
no viral cytopathic effect was seen in epithelial alveolar cells.
Only 1 of the 21 patients did not present interstitial changes
induced by viral disease. In this case, death was secondary to
pulmonary thromboembolism and bacterial pneumonia in a pa-
tient with laryngeal cancer.
All patients presented some degree of bronchiolar epithe-
lial necrosis and desquamation but it was extensive only in
patients with NB. Cytopathic effects of bronchiolar or alveolar
cells were observed in five cases. Microthrombi were observed
in six cases. Four patients had thrombosis of large pulmonary
arteries consistent with pulmonary thromboembolism (one
pregnant woman, two patients with cancer, and one obese
patient). In two patients, the DAD was associated with
In three patients (#1, 13, and 15), the histochemical search
for bacteria in the lung tissue was positive (Table 2).
Analysis of large airways showed epithelial necrosis, hyper-
plasia, and/or squamous metaplasia of the bronchial epithelium
and mucus gland ducts.
One patient had multiple bronchiolar and alveolar giant
cells. Immunohistochemical staining for herpes virus and re-
spiratory syncytial virus in this case was negative. No fungi or
acid-fast bacilli were found in the lungs of any patient.
Two patients (#15 and 20) presented metastatic cancer in the
nary changes. (A) Condensed, diffusely edematous and
hemorrhagic lung. Inset, a thrombus in a middle-sized
artery (arrow). (B) Large cartilaginous airway showing
intense epithelial squamous metaplasia, better visualized
in the inset. SM 5 squamous metaplasia. (C) Exudative
diffuse alveolar damage with numerous hyaline mem-
branes within alveolar spaces (arrowheads). (D) Extensive
lung hemorrhage as the predominant pattern of acute
lung injury. (E) Necrotizing bronchiolitis. The bronchiole
(Br) is filled with necrotic epithelial and inflammatory cells.
The adjacent alveoli (Alv) show fibrinous exudate rich in
inflammatory cells. (F) Necrotizing bronchiolitis, same
bronchiole as in E, Verhoeff staining. With the elastic
staining in dark gray, one can better appreciate the
extensive destruction of the airway wall. (G) Necrotizing
bronchiolitis (Br) with evident granulation tissue (Gt) in
the adjacent alveolar tissue indicating exudate organiza-
tion. (H) Cytopathic effect in alveolar epithelial cells
(arrow). Scale bars B–G: 100 mm; H: 25 mm.
Representative photomicrographs of pulmo-
Mauad, Hajjar, Callegari, et al.: Lung Pathology in H1N1 Infection 75
PCR. rRT-PCR testing for S-OIV was positive in the lung
tissue of 19 patients. In three patients (#2, 3, and 6), S.
pneumoniae DNA was also detected in lung tissue (Table 2).
In the pregnant patient, PCR testing for S-OIV in the placenta
Electron microscopy. Small vesicles containing one viral
particle enveloped by the pneumocyte II membrane were
observed at apical cytoplasm (Figure 2). Each vesicle was
roughly spherical and approximately 100 nm in diameter with
an electron-dense center. Other vacuoles of irregular shapes
and sizes containing more than one viral particle were also
found. In some particles, it was possible to distinguish the spikes
of surface glycoproteins as patches resembling a ‘‘picket fence’’
on the outer surface, a characteristic of the influenza virus
genera (19, 20).
Lung immunopathology. In
expressed in macrophages, bronchial and alveolar epithelial
cells, and endothelial cells. In S-OIV cases, there was a marked
TLR-3 expression in macrophages, in alveolar epithelial cells, in
vascular endothelial cells, and along the alveolar capillaries.
IFN-g was expressed weakly in alveolar macrophages and in
endothelial cells in control lungs. In infected cases, there was
a very strong expression in macrophages, alveolar epithelial
cells, and vessels. There was variable expression in the bronchial
epithelium. CD81T cells and granzyme B1cells were present
surrounding airways and in alveolar walls in control lungs. The
density of these cells was increased in infected cases, and the
cells tended to form small groups around small vessels and
bronchioles (Figure 3). TNF-a was expressed in alveolar
macrophages and bronchial and vascular smooth muscle in both
control and infected lungs.
There were no signs of direct virus-induced injury in any
examined organ other than the lungs. All patients had mild/
moderate kidney acute tubular necrosis. In four patients, there
was myoglobin pigment in the tubuli, and thrombotic angiopathy
was present in another patient. All patients presented atrophic or
nonreactive white pulp in the spleen. In the lymph nodes,
nonreactive follicles and sinusoidal erythrophagocytosis were
found (Figure 2). The liver showed erythrophagocytosis and
a few mononuclear inflammatory cells in the sinusoids in all
patients, and variable degrees of shock-related centrilobular
necrosis. Remarkably, the pregnant patient presented clinical
hepatic failure with massive hepatic necrosis. The placenta
presented signs of intrauterine hypoxia without signs of infection.
The fetus showed meconial aspiration in the lungs. No patients
presented histological signs of encephalitis, myocarditis, or
myositis. In most patients, there were also pathological changes
related to the underlying disease; this is not described here.
In this post mortem study, we report the pathological findings
from 21 patients with proven novel swine H1N1 infection who
died during the winter period of the 2009 pandemic in Sao
Paulo, Brazil. This is the first autopsy report detailing the
systemic pathology of this novel infection. Our data show that
the fatalities were related to extensive diffuse alveolar damage,
with variable degrees of pulmonary hemorrhage and necrotizing
bronchiolitis. This histological picture is associated with sus-
tained activation of the TLR-3 receptor, a large number of
cytotoxic cells, and marked expression of IFN-g in lung tissue.
monary changes and lung electron microscopy. (A) Spleen
with depleted white pulp (Wp) and expanded red pulp
(Rp). (B and C) Erythrophagocytosis in lymph node and
liver sinusoids, respectively (arrows). (D) Kidney acute
tubular necrosis. (E) Electron micrograph shows a small
vesicle (arrow) containing one viral particle in the inner
surface of a pneumocyte II. The particle is engulfed by
endocytosis at the cell surface. Spikes at particle surface
resemble a ‘‘picket fence’’ in the interior of the vesicle.
Arrowhead 5 cell membrane. (F) Electron micrograph
shows a cytoplasmic vacuole filled with numerous viral
vesicles. Scale bars A and D, 100 mm; B and C, 25 mm;
E and F, 200 nm.
Representative photomicrographs of extrapul-
76AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 181 2010