Using highly sensitive PCR, pan-viral microarrays, and deep sequencing technologies in a large, well-described cohort of patients with acute exacerbation of IPF and controls, we found that most cases of acute exacerbation of IPF had no evidence of an underlying viral infection. This suggests that viral infection is not a common cause of acute exacerbation of IPF.
Overall, we found viral nucleic acid in the BAL of 33% of patients with acute exacerbation of IPF; no viruses were found in samples from stable IPF controls. There were two rhinovirus-positive samples, one coronavirus-positive sample, and one parainfluenza virus–positive sample, suggesting that a small minority (9%) of acute exacerbations of IPF may be caused by occult infection with common respiratory viruses. Surprisingly, the most common virus detected in the BAL of acute exacerbation of IPF patients was TTV, which was present in 28% of acute exacerbation BAL samples. This finding was not unique to acute exacerbation of IPF because 24% of BAL samples from ALI controls were also TTV positive.
Two recent studies have commented indirectly on the possible role of occult viral infection in acute exacerbation of IPF. The first study performed gene expression microarrays on whole lung tissue from 8 patients who died of acute exacerbation of IPF, 23 patients with stable IPF, and 15 healthy controls (12
). The authors concluded that acute exacerbation of IPF was characterized by a pattern of enhanced epithelial injury and proliferation, but found no gene expression profiles indicative of a response to viral or bacterial infection. In a second study of 27 patients presenting with acute decline in fibrotic lung disease (13 of whom had confirmed acute exacerbation of IPF), 5 had antigenic or PCR evidence of viral infection (one parainfluenza virus, two HSV, and two CMV infections), three of which were missed on standard viral culture (11
Our study expands significantly on previously published reports. First, we take an unbiased approach to viral discovery using cutting-edge genomic methodology. It is the first study to do this in acute exacerbation of IPF. Our use of sequencing to confirm all suspected viruses rules out the possibility of spurious PCR results, a common pitfall of the technique. Second, our large cohort of well-defined patients with acute exacerbation with adequate controls allows for greater certainty regarding our conclusions. Third, we have identified an unexpected virus (TTV) that was associated with 33% of acute exacerbations, and that was absent in stable IPF.
The pathogenetic significance of TTV in acute exacerbation of IPF BAL is unclear. TTV is a nonenveloped single-stranded circular DNA virus that exists in a genetically diverse clade (22
). The virus seems to have broad tissue tropism because it has been detected in peripheral blood mononuclear cells (PBMCs) and bone marrow, spleen, liver, and lung (22
). Infection with TTV in the human population is worldwide, with prevalences of viremia ranging from 8%–80% depending on the population studied and detection methodology used. When only considering the hemi-nested PCR of the N22 region used in this study, rates of TTV DNA found in healthy blood donors range from 8.4%–12% (24
) and do not seem to correlate with the geographic location of the patients. Most infected subjects are asymptomatic, and to date efforts to link TTV viremia with any acute or chronic pathologic state have been unsuccessful (22
). Although there have been reports of TTV in the upper respiratory tract (nasopharynx and oral cavity) (26
), TTV has not been identified in BAL fluids. TTV has previously been detected in the serum of 12 (36%) of 33 Japanese patients with IPF. In this study, TTV appeared more frequently in cases that progressed to acute exacerbations, and TTV positivity was suggested to correlate with worse survival (27
). Our findings do not show a correlation between the presence of TTV in the serum and the presence of TTV in the BAL, or any correlation between serum TTV positivity and a diagnosis of acute exacerbation.
It is possible that de novo
TTV infection in the lung causes direct alveolar epithelial cell injury and acute respiratory worsening. If so, this process does not seem to be unique to acute exacerbation of IPF because we detected TTV at a similar frequency in BAL from patients with ALI. Although this does not exclude a potential role for TTV in the pathogenesis of acute exacerbation of IPF, it is also compatible with the idea that inflammation or injury in the lung may nonspecifically trigger local TTV replication, or may result in increased vascular permeability in the lung allowing circulating virus to enter the alveolar compartment. In the latter two cases, the presence of TTV would represent a consequence of lung inflammation rather than its cause. The idea that local TTV replication might be enhanced by underlying inflammatory signaling is supported by in vitro
studies of PBMCs from donors who are TTV negative (28
). These PBMCs were infected in vitro
with TTV, cultured with and without the presence of phytohemagglutinin, lipopolysaccharide, and interleukin-2, and then examined for evidence of TTV replication. In this experiment, TTV mRNA and replicative intermediates were only found in the stimulated PBMCs, consistent with an infection-amplifying role for inflammatory signaling.
The methodologies used in this study have unparalleled sensitivity for viral detection. The multiplex nested PCR is several fold more sensitive than virus culture and direct immunofluorescent tests, with the ability to amplify less than 10 copies of target nucleic acid (15
). For viral discovery, however, PCR is of limited use because it identifies only a priori
viral targets. Pan-viral microarray precludes the need for a preconceived list of targets, although even with its proved sensitivity, its benefit is dependent on the signal-to-noise ratio of the nucleic acid (19
). The use of deep sequencing to look further for evidence of viral infection in a high-risk subpopulation of patients with acute exacerbation therefore adds confidence to our results, because it produces an unbiased, high-resolution description of the microbial landscape of the sample tested. This technology has been used to identify novel viruses in human diarrhea, and to describe the microbiome of the distal gut (31
), but never in BAL (29
). In the current study, two samples subjected to deep sequencing were positive for known viruses by PCR and pan-viral array screening. Using an efficient and sensitive pipeline for sorting reads, these positive PCR findings were confirmed, and no additional viruses were detected in these or the other samples tested. Interpretation of these findings must be tempered by the fact that existing computational methods for recognizing potential viral genomes are imperfect, and may fail to identify novel agents with only limited homology to known viral genera. The same is true of array-based viral detection methods.
One important limitation of this study is the potential for false-negative results because of the timing of sample collection. BAL was performed early in the course of hospitalization, most commonly in the first 48 hours after admission, and the median time from symptom onset to sampling was 7 days. Importantly, no difference in the time from symptom onset to sample collection was found between virus-positive and virus-negative cases. The duration of replicating virus in BAL is largely unknown, and it is possible that a virus could have stopped shedding during this time. In this study, we have maximized our likelihood of detecting virus by obtaining BAL samples early after admission and using highly sensitive viral detection techniques.
In summary, this study used unbiased, highly sensitive genomics-based discovery methods to investigate the role of viral infection in a large, well-characterized cohort of patients with acute exacerbation of IPF. The results of this study suggest that most cases of acute exacerbation of IPF are not caused by viral infection. Future research into the etiology of acute exacerbation of IPF should confirm these findings, further investigate the role of TTV, and consider other possible occult complications (e.g., aspiration) that may cause acute respiratory worsening in these patients.