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Immunohistochemical assessment of respiratory viruses in necropsy samples from lethal non-pandemic seasonal respiratory infections
  1. Maria do Carmo Debur1,
  2. Sonia Mara Raboni2,
  3. Fabiane B Z Flizikowski3,
  4. Débora C Chong4,
  5. Ana Paula Persicote3,
  6. Meri B Nogueira5,
  7. Luine V Rosele5,
  8. Sergio Monteiro de Almeida5,
  9. Lucia de Noronha3
  1. 1Internal Medicine Postgraduate Program, Universidade Federal do Paraná, Curitiba, Brazil
  2. 2Virology Laboratory, Infectious Diseases Discipline, Universidade Federal do Paraná, Curitiba, Brazil
  3. 3Pathologic Anatomy Service, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, Brazil
  4. 4Department of Pediatrics, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, Brazil
  5. 5Virology Laboratory, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, Brazil
  1. Correspondence to Dr S M Raboni, Virology Laboratory, Department of Infectious Diseases, Hospital de Clínicas, Federal University of Paraná, R Padre Camargo, n 280, 2nd floor, room 203, Curitiba, Paraná 80060-240, Brazil; sraboni{at}ufpr.br

Abstract

Background/aim Acute respiratory infections are an important cause of childhood morbidity and mortality throughout the world, and viruses have often been reported to be an aetiological agent. This study aimed to identify respiratory viruses in paraffin-embedded samples of paediatric lung necropsy specimens, using immunohistochemistry on tissue microarray slides.

Methods Retrospective study in 200 lung tissue samples from children who had died from severe respiratory infections during 1985–2005. Immunoperoxidase assay was performed to detect the viruses that were most commonly associated with respiratory tract infections: influenza virus A (FLU A), influenza virus B (FLU B), respiratory syncytial virus (RSV), adenovirus (AdV) and parainfluenza virus (PIV) types 1, 2 and 3.

Results Viruses were detected in 71 (35.5%) cases. Most positive cases were observed in children younger than 6 months. In 42.3% of cases, only one virus was detected: 11 (36.7%) RSV; 7 (23.3%) AdV; 4 (13.3%) PIV2; 3 (10%) FLU A; 2 (6.7%) FLU B; 2 (6.7%) PIV3; and 1 (3.3%) PIV1. Co-infection with more than one virus was observed in 41 (57.7%) cases. No positive correlations were observed between the presence of viral antigens and seasonality of the infection, sex, age or histopathological findings.

Conclusions Non-pandemic seasonal respiratory viruses are involved in a significant number of deaths in paediatric patients; these findings highlight the importance of laboratory investigation of these agents in patients hospitalised with severe acute respiratory infections.

  • Respiratory viruses
  • tissue microarray
  • immunohistochemistry
  • paediatric lung necropsies
  • virology
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Introduction

Severe acute respiratory infections are an important cause of childhood morbidity and mortality throughout the world.1 2 Viruses have often been reported as aetiological agents of these infections.3–8 Respiratory virus infections in paediatric patients can cause clinical manifestations, which include complications of the upper and lower respiratory tract (URT and LRT), that often require hospitalisation.9–14 These infections are responsible for 1–3% of mortality among children younger than 5 years in industrialised countries and 10–15% of deaths in children in developing countries.13 15

The viruses most commonly associated with respiratory tract infections are influenza virus A (FLU A), influenza virus B (FLU B), respiratory syncytial virus (RSV), adenovirus (AdV) and parainfluenza virus (PIV) types 1, 2, 3, 4A and 4B.16–18 Other respiratory viruses have been described more recently, such as the human metapneumovirus19 and the human bocavirus.20

One pathogen was found to cause most paediatric LRT infections (60%), and viruses were the most common cause of pneumonia (39%), while bacterial infection only was found in 21%.21 Studies suggest that secondary bacterial or viral infections are relatively common following viral URT or LRT infections, although there is current debate about true primary and secondary infection.22 23 Aberle and collaborators,23 in a prospective study of children hospitalised due to respiratory infections, reported that, in most cases (57%), the presence of a single respiratory virus was detected. The authors estimated the frequency of dual viral infection to be 20–30%.23–26

The present study aimed to identify respiratory viruses in paraffin-embedded lung samples of necropsy specimens from children who had died from severe respiratory disease, using immunohistochemical assessment (IH) on tissue microarray (TMA) slides. These findings were also correlated with epidemiological data and the anatomopathological features of the injury.

Materials and methods

The study was performed with data from 200 necropsy samples from paediatric patients of Hospital de Clínicas of the Federal University of Paraná (HC-UFPR), from 1985 to 2005. The patients had died from severe respiratory infections. Necropsy samples from stillborn babies, children with primary or secondary immunodeficiency, and children submitted to haematopoietic stem cell transplantation were excluded from the study. These samples were selected from a database of the Paediatric Autopsy Data Bank, which contains results of 6362 perinatal and paediatric autopsies performed over a period of 40 years. The main underlying diseases reported for the selected patients were bronchopneumonia, pneumonitis, malnutrition, acute infectious gastroenteritis and congenital malformations. Septicaemia was the most common cause of death reported. The ethics review board of the HC-UFPR reviewed and approved the study (Register number 1099.138/2005-08; approved 30 August 2005).

The slides for IH assay were constructed using selected paraffin blocks containing two or more lung samples. Its corresponding slide stained with H&E was used for histological study and classification into two patterns according to the predominance of the histopathological findings: bronchopneumonia and interstitial pneumonitis.27

Representative pulmonary injury areas were marked on the H&E-stained section. The corresponding paraffin-embedded tissue block was punched with a hollow needle, and tissue cores 3 mm wide were removed and inserted into a recipient paraffin block in a specific space (TMA block).28 Therefore, multiple individual tissue samples were inserted into the same TMA block. Each TMA block included four samples from each patient and five to seven cases overall, positive and negative control tissues and standard tissue samples for intra-array orientation. Sections from this block were cut using a microtome, then mounted on a microscope slide (TMA slide), and analysed by IH. This technology enables simultaneous study of multiple tissue samples in the same slide.

Immunoperoxidase assay was used for IH with modifications as reported by Chong and colleagues.29 Briefly, specific monoclonal antibodies for antigen detection of AdV, RSV, FLU A, FLU B, PIV1, PIV2 and PIV3 of the ready-to-use Light Diagnostics Respiratory Viral Screen IFA Kit (Chemicon International, Inc, Temecula, California, USA) were used as primary antibodies.

Pulmonary biopsy specimens from patients in HC-UFPR with pneumonia caused by AdV, RSV, FLU A, FLU B, PIV1, PIV2 and PIV3 and/or a positive viral culture from nasopharyngeal aspirates were used as positive controls.29 30 The conventional technique, omitting the primary antibody and using RAM-11 antibody (Dako North America, Inc, Carpinteria, California, USA) as primary antibody, was the negative control.

Cases were considered positive if more than five cells were stained per high-power field in the bronchi/bronchiole epithelium and in the alveolar cells.

Statistical analysis to determine the association between viral respiratory infection and the variants gender, age, seasonality and histopathological standards was carried out using the χ2 test or Fisher exact test and the Pearson correlation test. The tests were performed on GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, California, USA). p<0.05 was considered significant.

Results

A total of 200 necropsy samples were selected for this study; the median age of the children was 7.0 months (ranging from 1 month to 14 years), and 109 (54.5%) were male. Viruses were detected in 71 (35.5%) cases. The median age of the infected patients was 7.0 months (ranging from 1 month to 13 years). Most positive cases occurred in cold months (table 1).

Table 1

Epidemiological and histopathological data for children who died from severe respiratory infections and the results of the viral-specific monoclonal antibody research

Table 1 shows the viruses detected and their correlation with age and histopathological standard. In 30 (42.3%) patients, only one virus was detected, which is detailed in table 2. Viral co-infection was observed in 41 (57.7%) cases. In 23 of these patients (56.1%), two or three viruses were detected, and, in 18 (43.9%), more than three viruses were detected simultaneously. These samples were retested for all the primary antibodies, and the findings were confirmed. The most frequently detected viruses in co-infections were RSV (87.8%), PIV3, FLU A and AdV. The histopathological standard for virus co-infection was the same as for single virus infection. Co-infection was highest in children younger than 6 months of age (44.2%). Bronchopneumonia was the main histopathological standard for both virus-negative and virus-positive samples (tables 1 and 2).

Table 2

Histopathological standard in lung necropsy samples due to single virus infection and its correlation with age

Destruction and cell necrosis of the pseudostratified columnar epithelium of the bronchi was observed on histological analysis. Specimens with no bronchopneumonia pattern showed mainly infiltrates of mononuclear inflammatory cells in the bronchial lumen. Diffuse alveolar damage with formation of hyaline membranes, in addition to necrotising bronchiolitis, was also found in some specimens, particularly from long-term hospital patients or virally co-infected patients. Later stages showed organising diffuse alveolar damage, fibrosis and alveolar epithelial regeneration (type II pneumocytes). These findings, associated with massive infiltration of neutrophils into alveolar air spaces, were often related to bacterial superinfection, particularly in long-term hospital patients. Capillary thrombosis followed by necrosis of the alveolar wall and intra-alveolar haemorrhage was observed in a few cases. The immunohistochemical stain was virus positive in the bronchial/bronchiole epithelial cells and alveoli cells of all 71 positive cases. Figure 1 in the supplementary online file shows the standard stain for each primary antibody used.

Discussion

Non-pandemic seasonal acute respiratory infections contribute significantly to the total death rate of children worldwide.31 32 However, very few studies have been conducted on this topic, and most have focused on reported morbidity, clinical care or hospital admissions.15 21 31 33–36 Most of these deaths were in children with pneumonia.11 37 Depending on the age of the child, 50–90% of LRT infections are caused by viruses.15 21 31 33 34 In the present study, viruses were detected in 71 (35.5%) lung necropsy specimens from children who had died from severe acute respiratory infections.

Histological changes in respiratory viral infections are non-specific. This analysis alone is insufficient to make a specific diagnosis, which typically requires supportive diagnostic tests such as virus isolation, antigen detection and serological studies, or a biopsy or autopsy tissue section confirmed by in situ hybridisation, IH or molecular techniques.30

In situ hybridisation or IH analysis of the URT or LRT has consistently demonstrated the presence of virus in tracheobronchial/bronchiole epithelial cells, mononuclear inflammatory cells and single alveolar epithelial cells, which are believed by some authors to represent sloughed bronchial epithelial cells, not alveolar lining cells. Taubenberger and Morens30 reported that the lack of antibodies for antigen detection of viruses in alveolar epithelial cells may be related to the time course of these infections. In this study, we observed bronchial epithelial cells, bronchiole epithelial cells and single cells in alveolar epithelium, positive by IH techniques.

Our results show that RSV is the most common virus associated with seasonal respiratory infections in children who need hospital care and treatment, particularly infants less than 6 months of age, followed by PIV3, FLU A, FLU B and AdV.11 12 18 37 38 RSV is the most common cause of pneumonia in infants and preschool-age children15 21 31 34 and also the most common virus found in children in our hospital, as previously reported.7

Two or more respiratory viruses were detected in 41 (57.7%) of the positive cases, which probably contributes to the severity of the disease.15 Co-infection with RSV was observed in the majority of mixed infections (87.8%), leading to the conclusion that co-infections with RSV lead to serious complications, and may cause death. It is important to point out that risk factors such as low socioeconomic status and low weight-for-age must be considered in the association of viral respiratory infection with mortality.39 40

In the 18 cases in which more than three viruses were detected, we recommend the use of other methodologies for virus investigation to confirm the identity of the detected viruses. Our recommendation is based on the fact that some authors report co-infections with up to three viruses in pneumonia cases,21 35 41 and we have detected more than three viruses in one patient who died from respiratory infection.

Determination of the aetiological agent in LRT infection is not easy because of the difficulty of obtaining appropriate specimens from the LRT.42 Pulmonary biopsies, transbronchial biopsies and pulmonary punches are invasive methods, with an inherent risk of complication.43 The study of pulmonary tissue, as well as cell cultures, and immunological methods to detect antigens have been reported to show high positivity and good representativeness of the infectious pulmonary focus.44–46 In our study, the sample composed of pulmonary tissue obtained from necropsy specimens made it possible to examine the infected area, as well as directing our immunological study and identifying the aetiological agent associated with the infection.

It is important to mention that we only looked at these seven viruses in this study because monoclonal antibodies for detecting other respiratory viruses, such as human rhinovirus, human metapneumovirus, human coronaviruses and human bocavirus, are not available in our region. Clearly, inclusion of the latter viruses in our study may have increased the number of positive samples, as well as shown the importance of the human rhinovirus as an agent in co-infection.

In the present study, we chose the IH assay because of easy visualisation and interpretation, with permanent availability of material, compared with the immunofluorescence assay in paraffin-embedded material, which has a signal of short duration, besides difficult visualisation.47 The ability to visualise the pulmonary tissue structures allowed us to adopt precise criteria for analysis of slides, avoiding false-positive results.48 TMA offers many benefits, such as the uniformity of staining reactions and time and reagent savings, and also helps to prevent necrotic and haemorrhagic areas which may complicate IH assessment.49–51 Viral antigen detection has been shown to be more sensitive and specific than cell culture isolation for respiratory virus diagnosis. However, comparing with molecular biology, it has been reported that antigen detection is positive in about 38% of cases, whereas PCR techniques are positive in more than 50% of cases, but these studies were performed with respiratory secretions.52 53 Nicholls et al54 described lung biopsy analysis by molecular methodologies for respiratory virus (FLU A, RSV and human coronavirus NL63) detection. Analysis of formalin-fixed tissues showed that PCR was a less-than-optimal method for detecting viral infections. As many respiratory viruses are RNA in type and the standard fixative of autopsy specimens is formalin, the extraction and detection of high-quality nucleotides suitable for PCR amplification remains a concern. So, the protocol used for respiratory virus detection in this type of material by PCR assay must be optimised to produce a reproducible assay with reliable results.

Bronchopneumonia was the main histopathological standard found, accounting for 78.5% of the total samples, followed by interstitial pneumonitis. Bronchopneumonia is the histopathological finding classically described in bacterial pulmonary infections.27 55 56 However, the present study, as demonstrated by other authors, shows that respiratory viruses—in isolation or in virus–virus co-infection—can be an aetiological agent of these findings,18 38 46 57 58 so histopathological standards cannot determine the aetiological agent associated with the infection. Consequently, laboratory diagnosis of the aetiological agent is important.57

Age distribution showed that most patients were less than 6 months of age, indicating that acute respiratory disease is one of the most common causes of death in these children.58–61 RSV is the most prevalent virus in children of this age in both developed and developing countries.62 63 In the present study, the highest number of positive cases among children younger than 6 months was for RSV (19 cases); however, this result was not statistically significant.

McFarlane and collaborators64 concluded that studies based on necropsy specimens compared with population studies produce more reliable data and probably less bias. Analysis of the dates of death of the patients showed no positive correlation between the presence of viral antigens and seasonality. Regardless of the viruses that circulate throughout the year, there were more deaths in cold (64%) than in hot (36%) months.

IH for the identification of respiratory virus in lung is a useful technique allowing standardisation of reactions and easy interpretation of the results. This study determined the most common viruses associated with child mortality from severe respiratory tract infection and emphasises the importance of early diagnosis of the aetiological agent for establishment of an appropriate treatment and/or implementation of prevention methods.

Take-home messages

  • Respiratory viruses were detected in one-third of paediatric necropsy samples.

  • There was no correlation between the presence of viruses and histopathological findings.

  • Tissue microarray offers many benefits such as uniformity of staining reactions and time and reagent savings.

References

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Footnotes

  • Competing interests None.

  • Ethics approval This study was conducted with the approval of the Hospital de Clinicas - Federal University of Paraná (Register number 1099.138/2005-08; approved 30 August 2005).

  • Provenance and peer review Not commissioned; externally peer reviewed.

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