American Journal of Respiratory and Critical Care Medicine

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 confirmed S-OIV infection are presented. These patients died in the winter of the southern hemisphere 2009 pandemic, with acute respiratory failure.

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 receptor (TLR)-3, IFN-γ, tumor necrosis factor-α, CD8+ T cells and granzyme B+ cells in the lungs was investigated by immunohistochemistry.

Measurements and Main Results: Patients were aged from 1 to 68 years (72% between 30 and 59 yr) and 12 were male. Sixteen patients had preexisting medical conditions. Diffuse alveolar damage was present in 20 individuals. In six patients, diffuse alveolar damage was associated with necrotizing bronchiolitis and in five with extensive hemorrhage. There was also a cytopathic effect in the bronchial and alveolar epithelial cells, as well as necrosis, epithelial hyperplasia, and squamous metaplasia of the large airways. There was marked expression of TLR-3 and IFN-γ and a large number of CD8+ T cells and granzyme B+ cells 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 increasing the understanding of the novel human influenza A (H1N1) infection.

Scientific Knowledge on the Subject

Most patients with H1N1 infection present flulike symptoms 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 associated 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 evidence of ongoing pulmonary aberrant immune response.

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 (13). 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 São Paulo. From June through October 2009, there were 494 confirmed cases of S-OIV infection 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, 810). 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). CD8+ T cells are important in the antiviral response to influenza via direct lysis or by the induction of tumor necrosis factor (TNF)-α and IFN-γ. Altered innate immune responses, excess of CD8+ T-cell cytotoxic responses, and hypercytokinemia are related to disease 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 hypothesize that a deregulation in antiviral immune response pathways could occur in the lungs and contribute to severity of the pulmonary manifestations.

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 Committee.


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 Sao Paulo.


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 until death.

The in vivo diagnosis of S-OIV infection was confirmed in nasopharyngeal swab specimens by using the real-time reverse transcriptase polymerase chain reaction (rRT-PCR) test, in accordance with guidelines from the Centers for Disease Control and Prevention (CDC) (15).

Tissue Processing

Tissue fragments were formalin-fixed, paraffin-embedded, and hematoxylin 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-α, IFN-γ (Santa Cruz Biotechnology, Santa Cruz, CA), CD8+ T cells (Dako, Carpinteria, CA) and granzyme B+ cells (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 tissue.

Pulmonary tissue from three cases was fixed for electron microscopy 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 previously healthy pregnant woman at 28 weeks of gestational age, did not have any identified comorbidity.




Sex, male(12/21)57
Age, yr
Median (range)34 (1–68)
Preexisting conditions
 Chronic cardiovascular disease(7/21)33
 Without comorbidity(5/21)24
 Chronic respiratory disease(2/21)10
 Chronic renal disease(2/21)10
 Kidney transplantation(1/21)5
 Bone marrow transplantation(1/21)5
Presenting manifestations
 Sore throat(2/21)10
Clinical features
 Respiratory failure requiring mechanical ventilation(21/21)100
 Admission in intensive care unit(16/21)76
 Acute respiratory distress syndrome(15/21)71
 Acute renal failure(9/21)43
 Acute renal failure with dialysis(4/21)19
 Shock needing vasopressors(13/21)69
 Treatment with oseltamivir(16/21)76
 Treatment with antibiotic(13/21)62
 Median of days from admission to death (range)6 (1–12)
 Corticosteroid therapy(12/21)57
 Clinically suspected bacterial pneumonia(6/21)29
Laboratory findings
 rRT-PCR in nasopharyngeal swab for S-OIV(21/21)100
 Bacteria isolation in culture*

Definition of abbreviations: rRT-PCR = real time reverse transcriptase polymerase chain reaction.

*Bacteria culture was obtained in bronchial aspirate samples in 9 patients.

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/FiO2 less 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 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 treatment 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 nasopharyngeal swabs.



Granulation Tissue Repair

Hyaline Membrane/Fibrinous Exudate

Alveolar Hemorrhage

Intraalveolar Edema

Interstitial Inflammation

Cytopathic Effect





Evidence of Bacterial Infection*
Histological pattern
 Necrotizing bronchiolitis with exudative DAD
  2ModerateModerateIntenseIntenseMildPresentPresentPresentNoneTissue PCR
  3MildModerateMildMildIntensePresentPresentPresentPresentEsophagus cancerTissue PCR
  4ModerateMildMildIntenseIntensePresentPulmonary fibrosisCulture of bronchial aspirate
  5MildModerateModerateModerateModeratePresentSAH, ventricular hypertrophy
  6ModerateModerateModerateMildModerateNoneTissue PCR
 Exudative DAD with intense alveolar hemorrhage
  7MildIntenseIntenseMildModeratePresentMyelofibrosis, bone marrow transplantationCulture of bronchial aspirate
  10MildIntenseMildMildKidney transplantation, chronic renal failure
  11MildMildIntenseMildModeratePresentDiabetes, coronary disease
 Exudative DAD
  12MildMildMildMildMildSAH, ventricular hypertrophy, diabetes
  14ModerateMildModerateMildPresentPresentPresentSAH, heart failure, chronic renal failure, systemic sclerosis
  15IntenseMildMildBowel cancerHistochemistry and culture of bronchial aspirate
  16MildMildMildIntenseModerateSAH, ventricular hypertrophy diabetes
  17MildModerateMildMildPresentSAH, ventricular hypertrophy, heart failure, diabetes
  18MildIntenseModerateModerateMildCongenital heart disease
  20IntenseMildModerateBreast cancer
 No virus-related pulmonary changes


Laryngeal cancer

Definition of abbreviations: DAD = diffuse alveolar damage; PTE = pulmonary thromboembolism; SAH = systemic arterial hypertension; PCR = polymerase chain reaction.

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.

Pulmonary Pathology

Macroscopic examination revealed heavy and consolidated lungs that were diffusely edematous with variable degrees of hemorrhage (Figure 1).

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, however, 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 patients, 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. Parenchymal inflammation in these patients was severe and predominantly 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 patient with laryngeal cancer.

All patients presented some degree of bronchiolar epithelial 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 alveolar necrosis.

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, hyperplasia, 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 respiratory 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 lungs.


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 was negative.

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 control lungs, TLR-3 was 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-γ 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. CD8+ T cells and granzyme B+ cells 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-α was expressed in alveolar macrophages and bronchial and vascular smooth muscle in both control and infected lungs.

Extrapulmonary Findings

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 sustained activation of the TLR-3 receptor, a large number of cytotoxic cells, and marked expression of IFN-γ in lung tissue.

The studied patients presented a progressive and rapidly fatal form of the infection, characterized by a severe impairment of respiratory function. All patients required mechanical ventilation and developed multiple organ failure. More than half of our patients were between 30 and 59 years of age, similar to the age distribution reported in other series (1, 8, 21, 22). In a Mexican series of S-OIV cases (8), of 12 patients who needed mechanical ventilation, 7 died.

Previous data on S-OIV infection in humans have shown that the majority of affected patients are healthy individuals who present with fever, cough, and myalgia occurring in nearly 100% of cases (8, 22). In our series, most patients with a fatal form of the disease presented with dyspnea, with fever and myalgia being less frequently present (23).

The cause of death in all patients was extensive involvement of the lungs. Twenty patients had severe diffuse alveolar damage with varying degrees of alveolar hemorrhage, necrotizing bronchiolitis, and tracheobronchitis. Histologically, we could not detect direct signs of virus-induced disease in the other examined organs or in the placenta or fetus of the pregnant patient.

The association between influenza and bacterial coinfections has been related to increased morbidity and mortality in earlier pandemics (24). In the novel S-OIV infection, this also seems to be the case. Recently, the CDC reported that 29% of fatal cases in the United States presented at least one bacterial coinfection (10 cases with S. pneumoniae, 6 with Streptococcus pyogenes, and 7 with Staphylococcus aureus) (25). In our report, we found evidence of bacterial coinfection in 8 out of 21 patients (38%). In six patients bacteriological analysis by culture of bronchial aspirate and/or tissue PCR revealed S. pneumoniae as the etiological agent. Influenza virus and pneumococcus are the most common pathogens associated with dual infections. It has been suggested that the pathways and intermediate signaling molecules are similar in both infections, creating an opportunity for either interference with or augmentation of the immune response during dual or sequential infection (24).

There are previous autopsy reports on the 1918, 1957, and 1968 pandemics, as well as on the deaths caused by avian influenza. Notably, the histological changes of the novel S-OIV infection are similar to what has been previously described in severe cases of influenza infection, such as diffuse alveolar damage, alveolar hemorrhage, necrotizing bronchiolitis, and the histological correlates of the multiple organ dysfunction syndrome, such as kidney tubular and hepatic centrolobular necroses (2631). It seems that histological examination alone is not enough to explain the different mortality rates observed in the different pandemics during the last and present centuries.

However, the identification of three distinct patterns of lung involvement among the present cases may be of clinical relevance. Patients with necrotizing bronchiolitis had a more severe neutrophil-predominant inflammatory exudate compared with the others. Previous reports on the pathology of influenza virus have indicated that the presence of many neutrophils in the lung tissue strongly suggests a bacterial coinfection (26). Indeed, five of the six patients with necrotizing bronchiolitis had evidence of bacterial infection, suggesting patients with NB are more prone to developing coinfections. As confirmed in previous series of severe H1N1 infection (10), most of our patients presented comorbidities. Interestingly, none of the five patients without comorbidities had hemorrhagic lung involvement. Furthermore, in the group of patients with severe alveolar hemorrhage, no alveolar cell viral cytopathic changes could be detected. These findings suggest that severe alveolar hemorrhage is associated with comorbidities, such as chronic cardiovascular disease and coagulopathies, conditions that predispose the patients to increased alveolar pressure and bleeding.

Only one patient in this series was pregnant. It is interesting to note that this patient had the most severe lung injury, with an extensive necrotizing bronchiolitis associated with DAD with multiple areas of alveolar necrosis, as well as a massive hepatic necrosis (patient #1 in Table 2 and Figure 1).

Nine patients developed acute renal failure, and four required dialysis. All patients presented mild to moderate forms of acute tubular necrosis. In avian influenza cases, the virus has been detected in the kidneys (32). Although we have not searched for virus in kidney samples, we could not find direct evidence of virus-induced renal lesions. We have not performed an extensive and detailed analysis of the skeletal muscles in the autopsies, but it is interesting to note that in four patients, myoglobin pigment was found in the kidneys, reflecting that some degree of skeletal muscle injury might have occurred. In previous reports of fatalities in the influenza pandemics and in avian H5N1 infections, extrapulmonary viral disease has been reported, such as sporadic cases of central nervous system disease, myocarditis, and myopathy (33).

In our study, changes in organs other than the lungs were mainly secondary to multiple organ failure. Although there were no histological signs of extrapulmonary virus–induced disease, we do not present any data on the presence of viral RNA/protein in other organs. However, sound evidence for replication of influenza virus in extrarespiratory tissues is still missing (33).

This report has some important limitations. The retrospective nature of the work limited the number of clinical correlations that could be performed. Because we believed it was important to provide information on the pathology of this new infection in a timely manner, our lung immunopathological studies are based on qualitative analysis, and we have therefore not analyzed systematically differences among the different lung histological patterns.

The pathogenesis of severe lung injury related to influenza infection in humans is poorly understood, and no information on human S-OIV infection is yet available. The primary antiviral mechanism involves activation of the TLRs and of cytotoxic CD8+ T cells (34). TLR-3 activation is an important signaling pathway for the recognition of dsRNA and for the triggering of antiviral responses (35). Children with severe influenza infection present defective responses to TLR-3 ligands by producing lower cytokine levels, and TLR-3 agonists are being proposed as a therapeutic tool in severe infections (35, 36). Conversely, experimental studies in TLR-3−/− mice have shown that reduction of TLR-3–mediated inflammatory response reduces the clinical manifestations of influenza-induced pneumonia (37) and protects from agonist-induced changes in lung function (38).

The antiviral mechanisms related to the action of CD8+ T cells are via direct lysis of infected cells or by production of inflammatory cytokines. However, CD8+ T cells also contribute to tissue injury in the course of a viral infection. Immunopathology caused by CD8+ T cells is more apparent in cases of high viral doses, when the T-cell response does not control viral loads (39). The drastic decrease in CD8+ T lymphocyte infiltration concomitant with prolonged survival in the TLR-3−/− mice suggests that a dysregulated TLR-3–dependent CD8+ T-cell response may lead to sustained lung injury in severe influenza infection (37).

An influenza virus–induced “cytokine storm” is believed to be involved in the pathogenesis of severe forms of influenza (40). The high circulating levels of cytokines, such as IFN-γ and TNF-α, are associated with the erythrophagocytosis observed in severe infections and are also present in our cases. In experimental models of influenza infection, the role of TNF-α and, to a lesser extent, of IFN-γ in lung immunopathology has been described (39). In our samples, there was variable lung expression of TNF-α. It was recently described that in swine influenza virus H1N2 infection, the expression of TNF-α in the lungs of infected pigs peaked in the first days of the infection and gradually decreased. If the same happens in humans infected with S-OIV, this could help explain our findings (41).

Taken together, our results point out that in fatal S-OIV infection, viral overload leads to altered innate immune responses, with sustained TLR-3 activation and consequently enhanced inflammation with high numbers of CD8+ T/granzyme B+ cytotoxic cells and local production of IFN-γ.

In summary, autopsies on patients who died with this novel infection have shown that the respiratory failure observed in patients with H1N1 is related to severe DAD and aberrant immune responses. In a world scenario where few autopsies are performed, this study underscores the extreme usefulness of this procedure, which in this case, combined with virologic and immunologic analysis, can shed some light on our understanding of the novel human influenza A (H1N1) infection.

The authors thank Dr. Regina Schultz, Dr. Rafael Moura, and the pathologists of the Sao Paulo Autopsy Service for performing autopsies and assisting with histological examination. We are indebted to Instituto Adolpho Lutz–Sao Paulo for performing PCR in the lung samples.

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Correspondence and requests for reprints should be addressed to Thais Mauad M.D., Ph.D., Department of Pathology, São Paulo University Medical School, Av. Dr. Arnaldo, 455 room 1155, CEP 01246-903 São Paulo SP, Brazil. E-mail:


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