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Print version ISSN 1679-4508
Einstein (São Paulo) vol.10 no.3 São Paulo July/Sept. 2012
Roberta MarchioriI; Carla Sakuma de Oliveira BredtII; Marcos Menezes Freitas de CamposI; Fábio NegrettiI; Péricles Almeida Delfino DuarteI
IHospital Universitário do Oeste do Paraná - HUOP, Cascavel (PR), Brazil
IIUniversidade Estadual do Oeste do Paraná - UNIOESTE, Cascavel (PR), Brazil
OBJECTIVE: To analyze the histopathological lung findings of four fatal cases of the 2009 H1N1 influenza pandemic and their correlation with clinical and epidemiological characteristics.
METHODS: Descriptive data from medical records of four patients who died in the Intensive Care Unit of a university hospital in 2009. Nasopharyngeal aspirate specimens were collected from the patients and were analyzed by real-time polymerase chain reaction. Lung biopsy was performed post mortem; a score of intensity for pathological changes was applied.
RESULTS: Three patients had positive real-time polymerase chain reaction (although all of them had a clinical diagnose of influenza H1N1). The main histopathological changes were: exudative diffuse alveolar damage with atelectasis; varying degrees of alveolar hemorrhage and edema, necrosis and sloughing of the respiratory epithelium in several bronchioli; and thrombus formation. One of the patients (the pregnant one) presented histopathological findings of cytomegalic inclusion.
CONCLUSION: The pulmonary histopathological findings in patients with fatal 2009 H1N1 influenza pandemic disclosed intense alveolar damage and hemorrhage and severe bronchiolitis. A co-infection with cytomegalovirus was described in the pregnant patient.
Keywords: Influenza A virus, H1N1 subtype; Pandemics; Intensive care units
Influenza pandemics are unforeseeable and associated to high morbidity and mortality, as well as to economic and social turmoil(1). Influenza viruses have a strong capacity for mutation; their antigenic variation, frequently make the virus unrecognizable to the immunological system and every new subtype becomes a threat, being able to cause a serious epidemic or even a new pandemic(2).
In 2009 a new strain of A/H1N1 influenza virus, with a blend of genic segments non previously identified either in humans or animals provoked an influenza pandemic(3). Firstly identified in Mexico and in the United States, it spread globally in few weeks via international flights(4,5).
Brazil was particularly hit: more than 45 thousand people were infected in the epidemiological year of 2009, when 2052 deaths occurred. The country south and southwestern regions were the most affected(6,7). In Paraná state, in 2009, 63 patients with the diagnose of H1N1 influenza were admitted in 11 Intensive Care Units (ICU) in a period of 45 days presenting a high mortality rate(8). In 2010, another 1623 cases of influenza A/H1N1 still occurred, according to the data of the Secretaria da Saúde do Paraná(9).
To analyze the histopathological lung findings of four fatal cases of the 2009 H1N1 influenza pandemic and their correlation with clinical and epidemiological characteristics.
This is a retrospective study reviewing the medical records of four patients clinically diagnosed with H1N1 virus infection that were admitted to the ICU of Hospital Universitário do Oeste do Paraná (HUOP) of Cascavel city, in 2009, had death as outcome and were submitted to lung biopsy.
The following data were collected: gender, age, origin of the patients, the presence of co-morbidities, clinical follow-up, laboratory tests and ventilator parameters.
The four patients belonged to the female gender. One of them (patient A) was an AIDS patient; one other case (patient B) presented a story of neurodevelopmental retardation; a third patient (patient C) was pregnant with a gestational age of 34 weeks; and the last patient (patient D) had heart insufficiency, liver dysfunction and chronic kidney disease. All patients came from cities of the western region of Paraná state, near the Paraguayan and Argentinian frontiers.
Samples of nasopharyngeal aspirate were analyzed at the Central State Laboratory in Curitiba (Paraná) by the real time polymerase chain reaction (RT-PCR) method. The SuperScriptTM III Platinum® One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, EUA) kit was used to process the samples.
The patients were submitted to post-mortem lung biopsy. The samples were fixed in 10% buffered formalin, embedded in paraffin blocks, and then stained by hematoxylin-eosin, Grocott methenamine-silver, and Ziehl-Neelsen techniques for fungal and acid- fast bacilli identification. Sample culture was not performed.
A histological semi-quantitative evaluation was made by attributing a score from 0 to 3 (0=absent; 1=light; 2=moderate; 3=severe) for the following histological parameters: granulation tissue, hyaline membrane, alveolar hemorrhage, intra alveolar edema, interstitial inflammation, cytopathic effect, perivasculitis, microthrombus, thromboembolism, besides highlighting the presence of bacteria, acid-fast bacilli or fungus (criteria adapted from Mauad et al.(10)). After the score was attributed by the pathologist in a blind way, the total sum was registered showing a maximum score for each sample.
The present study was approved by the Research Ethics Committee of the Universidade Estadual do Oeste do Paraná (UNIOESTE).
The patient's mean age was 35.5 years (21 to 65 years).
A post mortem surgical removal of lung specimens was performed in all patients. The microscopic findings are shown in table 1.
The main clinical findings and their frequency were the following: fever and dyspnea (present in 100%); myalgia (in 50%); cough (in 75%); vomits (in 25%). None of the cases presented diarrhoea. All cases needed mechanical ventilation. Acute kidney insufficiency developed in two cases but dialysis was not necessary.
All patients that were RT-PCR positive presented the acute respiratory distress syndrome (ARDS). This diagnose was established by the presence of a radiological bilateral lung infiltrate and the relation between arterial oxygen partial pressure/fraction of inspired oxygen in inspired air (PaO2/FiO2) <200.
Patient A presented hemoptysis and had low platelet count since her hospital admission. Patient C, the pregnant one, presented the same complication but had a normal platelet count. All patients were treated with oseltamivir for at least five days with the recommended dosage according to the Ministry of Health protocol. Corticotherapy was also used in all patients as well as vasoactive drugs. Table 2 shows the clinicopathological data of the four patients.
All patients presented various degrees of alveolar damage. Severe alveolar hemorrhage occurred in one patient, moderate in another, and light in a third. Pneumonitis was found in various degrees, moderate interstitial inflammation in one patient and severe in three of them. Two patients presented low grade perivasculitis. The presence of microthrombus and thromboembolism was severe in two cases and moderate in one. No cytopathic effect was found nor infection either by acid-fast bacilli or fungus (Table 1).
Patient B presented a severe degree of alveolar hemorrhage and edema receiving the highest score, and clinically had the worst respiratory clinical course.
Figure 1 shows the histopathological findings of one of the patients (patient C) who was RT-PCR positive. Her bronchoalveolar epithelium showed a cytomegalic inclusion which was compatible with a concurrent cytomegalovirus infection.
Patients A and B showed severe perivasculitis and thromboembolism. All cases presented severe necrosis and scaling of the respiratory epithelium in various bronchioli. A few alveoli were expanded and the pulmonary tissue among these alveoli showed atelectatic alveoli.
All cases presented severe acute respiratory insufficiency related to the 2009 influenza pandemic virus H1N1, were admitted to the ICU and needed orotracheal intubation.
The four patients belonged to the female gender and the three confirmed cases occurred in young adults. In a Brazilian study involving 21 patients with confirmed 2009 influenza pandemic the mean age was 34 years(10).
Co-morbidities were possibly related to the disease severity and the patients' worst course. In a USA study in 2009, involving 272 patients, presenting a confirmed H1N1 diagnose, 73% had at least one co-morbidity(3,11).
Among the patients of the present study, there were no obese ones, different from what was found in patients of a ICU cohort from Australia and New Zealand, in 2009. From the 722 patients studied, 601 had available data for weight and 28.5% had body mass índex >35; while in California, among 268 hospitalized patients, 58% were obese(12).
All patients in this study had fever, dyspnea and severe respiratory insufficiency that required mechanical ventilation. Myalgia occurred in two patients and vomiting in one. None had diarrhoea. In the Mexican epidemics cough, fever and dyspnea were the prevailing clinical signs(13). In an American study involving 642 patients, dyspnea was not reported, 94% cases presented fever and 92% had cough. However, in this American study not all patients were hospitalized(14).
The patients' fatal course in the present study may be explained by the risk factors that were present favoring the worst prognosis, such as pregnancy and immunossupression (the AIDS patient).
As related to the laboratory tests (Table 2), lymphopenia was present in all 2009 H1N1 influenza pandemic patients and a low platelet count was found in patient A, probably related to AIDS. There was an important high level of lactate dehydrogenase (LDH) in the three confirmed cases and a high level of creatine phosphokinase (CPK) in two cases. Patient B presented levels over 1.000U/l LDH, with the worst histological picture and respiratory course as compared to the others implying in a severe degree of pulmonary injury.
Patient A was an AIDS case presenting severe respiratory insufficiency and an associated clinical hypothesis of a pneumocystic infection which could explain the considerable increase in the LDH level.
Increased CPK level over 1.000U/l was found in the pregnant patient. High CPK and LDH levels as well as lymphopenia were described in Mexican patients with respiratory insufficiency due to the 2009 influenza pandemic H1N1(13).
The confirmed cases presented various degrees of alveolar hemorrhage, edema and diffuse alveolar damage on histopathology, similarly to previous influenza pandemics(15). Patient B presented the worst respiratory course obtaining also the worst pathological score according to the histological classification, besides presenting ARDS (the same fact that occurred with the other two confirmed cases). She also required prone positioning on two occasions, probably associated to the severe alveolar edema and hemorrhage as shown by the biopsy. In order to improve ARDS, a few mechanisms must remain active such as the reabsorption of pulmonary edema from the alveolar spaces. A decrease in the capacity of the alveolar epithelial barrier to remove liquids was demonstrated in patients with acute lung injury(16). Some studies have demonstrated that the alveolar deficiency in transporting liquids is directly related to the severity of the clinical picture in patients with ARDS(17,18). It was also observed in mice infected by influenza A virus that this virus promotes a clearance blockage of alveolar fluid directly compromising gaseous exchanges(18) .
The prevalence of infection by the pandemic H1N1 virus in pregnancy has been described in the literature as being between 7% and 9%(3,11). The pregnant patient (patient C) presented histological findings compatible with the presence of cytomegalovirus in the bronchoalveolar epithelium, a concurrent infection with the H1N1 disease, which may have contributed to the severe interstitial inflammation found in this case. Cytomegalic pneumonitis is an uncommon complication and its diagnose was based on the direct visualization of a cytomegalic inclusion and its cytopathic effect on the pulmonary tissue(19). The description of this co-infection was not found in the literature. It is reported that the majority of pregnant women have been previously infected by cytomegalovirus; however, approximately 15% of them are susceptible to primary infection during pregnancy(20,21). In this patient, microthrombus and moderate thromboembolism (according to the previously referred score) were found in the histological preparation. This finding could be related to a gestational hypercoagulable state but also to 2009 H1N1 virus itself, as reported by the Pathology Department of São Paulo University (USP)(10,22) .
Patient A, an AIDS carrier, presented severe perivasculitis and thromboembolism that may be related to the extensive pleural effusion (caused by hydrostatic pressure changes) that needed thoracic closed drainage. Besides, a severe interstitial inflammation was present, as in patient C the one suspected to have a concurrent cytomegalovirus infection. However, in patient A, it might have been the result of a clinically suspected infection by Pneumocystis jirovecii despite the negative search in the tracheal secretion and the fact that P. jirovecii was not visualized on the histological preparations that were performed.
Patient D was PCR negative for H1N1 in nasopharynx and oropharynx aspirates. However, a specimen from the inferior respiratory tract was not collected, which could bring a weighty evidence for the diagnose as it is described in the literature(23). Up to 10% of severe H1N1 influenza cases (admitted to ICUs) showing a RT-PCR positive test in the tracheal secretion have negative exams in the nasopharynx and oropharynx secretions(24). Nevertheless, the patient's clinical course differed from the other ones. She presented leukocytosis during her entire hospital stay; she also presented acute respiratory insufficiency but it was only on the third admission day that she required orotracheal intubation and did not course with ARDS (the other patients needed mechanical ventilation immediately before ICU admission).
ICUs patients when RT-PCR positive present a worse clinical outcome when compared to those with a negative result(8). So it is possible to speculate that the H1N1 infection in this patient (older and with co-morbidities) could not have been so severe but the outcome was inauspicious because of her age and the co-morbidities. The main histological lung change in this patient was interstitial inflammation which is not specific. Alveolar hemorrhage and the presence of thrombus that were found in the other patients with positive tests were absent in this case.
Patient B had the worst histological score, but she did not have any co-morbidities that could justify such an ominous course; so maybe a more virulent viral mutation could have infected this patient being capable of promoting the histological changes that were found with greater intensity.
This article has limitations, including the fact that it is a retrospective one. Cultures to identify bacteria were not performed in the patients' respiratory specimens because of technical problems in the laboratory as well as cultures were not performed in the lung biopsies because the specimens were processed in formaldeyde. The case that had a probable concurrent infection with cytomegalovirus did not have all laboratory tests for its confirmation, because such an hypothesis was not raised by the time the patient was being cared for.
These four fatal cases of pandemic influenza H1N1 with lung histopathology showed as the main findings exudative alveolar damage with alveolar atelectasis; various degrees of alveolar hemorrhage and edema; necrosis and scaling of the bronchioli epithelium. A rare concurrent infection with cytomegalovirus in one patient was also described.
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Corresponding author: Received on: Jan 11, 2012 Study carried out at Hospital Universitário do Oeste do Paraná - HUOP, Cascavel (PR), Brazil.
Avenida Tancredo Neves, 3.224 - Santo Onofre
Zip code: 85806-470 - Cascavel (PR), Brazil
Phone: (55 45) 3321-5151
Accepted on: May 2, 2012
Conflict of interest: none.
Received on: Jan 11, 2012
Study carried out at Hospital Universitário do Oeste do Paraná - HUOP, Cascavel (PR), Brazil.