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Department of Pediatrics and Virology Section and Retrovirus Center, Department of Experimental Pathology, University of Pisa, Pisa
Department of Pediatrics, University of Verona, Verona, Italy
Fifty-nine children with well-controlled, mild to moderate persistent asthma were studied for the presence and load of torquetenovirus (TTV) in nasal fluid. Rates of TTV positivity and mean nasal TTV loads were not dissimilar to those observed in the general population and in a group of 30 age- and residence-matched healthy control children without a history of asthmatic disease. However, in the children with asthma, 3 important indices of lung functionforced expiratory flow (FEF) in which 25% and 75% of forced vital capacity (FVC) is expired (FEF25%75%), forced expiratory volume in 1 s/FVC, and FEF25%75%/FVCshowed an inverse correlation with nasal TTV load. Furthermore, signs of reduced airflow were more frequent in the children with asthma who had high nasal TTV loads (6 log10 DNA copies/mL of nasal fluid) than they were in those who had low nasal TTV loads (<6 log10 DNA copies/mL of nasal fluid), despite similar therapy regimens. In contrast, the control children showed no associations between nasal TTV load and the spirometric indices. Levels of eosinophil cationic protein in sputum were also greater in the children with asthma who had higher nasal viral burdens than they were in those who had lower nasal viral burdens. These findings are the first report of TTV infection status in children with asthma and suggest that TTV might be a contributing factor in the lung impairment caused by this condition.
Torquetenovirus (TTV) is a member of the genus Anellovirus, which was recently established (but has not yet been classified within any viral family) to accommodate a number of small viruses with single-stranded circular DNA genomes that could not be classified within the family Circoviridae [1]. Early studies after its discovery in 1997 focused on TTV as the possible causative agent of cryptogenetic hepatitis, but the observation that chronic TTV viremia is widespread among the general population worldwide has led to considerable uncertainties about what possible pathogenic potential it may have, if any [2, 3].
Recently, by studying children 2 years old who had acute respiratory diseases, we and others demonstrated that the respiratory tract is a site of primary infection and continual TTV replication [4, 5]. Although no evidence was obtained to suggest that TTV is a direct cause of respiratory disease, mean TTV loads in plasma and nasal fluids were considerably higher in children with bronchopneumonia than they were in those with milder illnesses [4]. Furthermore, TTV loads were negatively related to the percentages of circulating CD3+ and CD4+ T cells and were positively related to the percentage of circulating B cells, suggesting that TTV might have immunomodulatory effects [6]. Finally, the presence and load of TTV in plasma were found to correlate with the levels of eosinophil cationic protein (ECP) in serum [7]. Because it has been proposed that high serum ECP levels in young children predict an increased likelihood of developing airway hyperreactivity, wheezing illnesses, and asthma at later times [811], we hypothesized that TTV infection might represent a heretofore unrecognized inducer or, more likely, facilitator in the pathogenesis of these extremely common afflictions [7].
Prompted by the above findings, in the present study we assessed the status of TTV infection in the upper respiratory tract of a well-characterized group of children with well-controlled, mild to moderate persistent asthma and examined whether TTV infection status was related to lung function. Our results suggest that TTV might indeed contribute to the pathogenesis of asthma.
PARTICIPANTS, MATERIALS, AND METHODS
Study populations.
The asthmatic population consisted of 59 children with well-controlled, mild to moderate persistent asthma (51 boys and 8 girls; mean ± SD age, 11.5 ± 2.2 years; age range, 716 years) admitted for a medical examination to the Department of Pediatrics, University Hospital of Pisa, from January 2002 to July 2003. Asthma was diagnosed in accordance with the recommendations of the Global Initiative for Asthma [12, 13]. All children were born after at least 37 weeks of gestation, weighed >2700 g at birth (mean weight, 3547 ± 422 g; weight range, 27704250 g), were nonsmokers, and were serologically negative for hepatitis B virus surface antigen and antibodies to hepatitis C virus and HIV. None had a history of receiving antiviral drugs, blood, or blood products. At the time of testing, all of the children were asymptomatic, having had no signs of acute asthma or exacerbations for at least 2 months, and were receiving long-term treatment with inhaled corticosteroids at doses ranging from 200 to 1000 g/day. Twenty-one of the children with asthma were also receiving long-acting 2-agonists (19 salmeterol and 2 formoterol), and 15 were also receiving montelukast. Thirty age-matched healthy children served as the control subjects; these children lived in the same area as the ones with asthma but had a negative history of asthma, atopy, and wheezing, as determined by questions that were based on recommended questionnaires [11]. All enrolled participants were physically examined and were assessed for lung function by spirometry. On the same day, nasal fluids were collected and stored until being tested in a blinded fashion for the presence and load of TTV. The children with asthma also received concomitant skin-prick tests with a panel of standardized allergen extracts [14] and provided sputum specimens. Informed consent was obtained from the parents of all children who provided specimens.
Lung function tests.
Combined partial and maximal forced expiratory flow (FEF) volume maneuvers were performed by use of MasterScreen Body equipment (Jaeger). Supervised by a single experienced examiner (M.P.), measurements were taken with the child seated and included forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), and FEF where 25% and 75% of FVC was expired (FEF25%75%). All children performed a minimum of 3 forced expiratory maneuvers, starting in the maximum inspiratory position, until at least 2 comparable flow-volume curves were obtained (i.e., with FEV1 and FVC values differing by no more than 5%) [15]. The results are expressed as a percentage of the normal values predicted for children of the same age, height, and body surface area, according to reference values [16].
Specimen collection and processing.
Nasal swabs were collected and rapidly transferred to the clinical virology laboratory of the Department of Experimental Pathology, University of Pisa, where they were immediately processed, as described elsewhere [4]. Briefly, the nasal-secretion volume was measured, and then secretions were diluted in PBS and centrifuged. Cell-free nasal fluid was aspirated and stored in aliquots at -80°C until use. For sputum induction, the children were premedicated with 200 g of inhaled salbutamol, to inhibit possible bronchoconstriction, and were then exposed via inhalation to a 3.5% NaCl aerosol solution generated by an ultrasonic nebulizer (Omron U1; Omron Healthcare) for a total of 20 min. FEV1 was recorded before and 10 min after salbutamol administration and then every 5 min during saline inhalation. The nebulization was stopped when FEV1 decreased by 20% from baseline value or when symptoms of bronchoconstriction occurred [17]. The children were instructed to rinse their mouths, to minimize salivary contamination, and were encouraged to cough deeply into a sterile plastic container. Valid sputum specimens were, however, produced by only 32 children. These were processed within 2 h of collection by a method that has been described elsewhere [18]. Briefly, after treatment with 0.1% dithiotreithol and complete homogenization, the sputum specimens were filtered to remove cell debris and mucus, and the total number of nonsquamous cells was counted manually by use of a hemocytometer (Heinz-Herenz). After centrifugation, the sputum supernatants were stored at -70°C for later ECP analysis, and resuspended cell pellets were used for cytocentrifuge slide preparation. Differential cell counts were performed on a minimum of 500 nonsquamous cells and are expressed as percentages of these cells. Sputum ECP levels were determined by a commercial radioimmunoassay (UniCAP 100; Pharmacia and Upjohn), in accordance with the manufacturer's instructions. All sputum ECP levels were measured in duplicate; the intra- and interassay coefficients of variation were <3%, and the lower limit of detection was 2 ng/L.
TTV detection and quantification.
Viral DNA was extracted from 200 L of nasal fluid by use of the QIAamp DNA Mini Kit (QIAgen). Presence and load of TTV were determined in triplicate by a universal TaqMan real-time polymerase chain reaction (PCR) assay targeted to a highly conserved segment of the noncoding region of the viral genome. This assay is potentially capable of amplifying all of the genetic forms of TTV hitherto recognized at a lower limit of detection of 1000 DNA copies/mL of nasal fluid [4, 6, 7] but, as determined by sequence data, does not amplify the other anellovirus, which is known as "torquetenominivirus" because of its smaller genome [19]. The procedures used for the quantification of copy numbers and the evaluation of specificity, sensitivity, intra- and interassay precision, and reproducibility of the assay have been described elsewhere [4, 20].
TTV characterization.
Nasal specimens found to be virus positive by the universal PCR assay were amplified by 5 distinct PCR protocols, each of which was specific for a TTV genogroup and had a lower limit of detection of 4000 DNA copies/mL of nasal fluid. The specificities and sensitivities of these assays, as well as the modification of certain primer sequences introduced to expand the breadth of detection, have been described elsewhere [4, 6, 7, 21]. All specimens were tested at least in duplicate.
Rhinovirus detection.
Because the children with asthma were free from exacerbations at the time of testing, they were not systematically examined for other common pathogens that have been implicated in the pathogenesis of exacerbations [22, 23]. However, all of the children were screened for nasal carriage of rhinoviruses, to assess for a possible association with TTV presence and load. The method used consisted of an in-house nested reverse-transcriptase PCR assay targeted to the conserved noncoding region of the viral genome [24].
Statistical analyses.
Statistical analyses of TTV loads were performed on log10-transformed values, to approximate a normal distribution, and with the children without detectable TTV considered to have a viral load of 3.0 log10 DNA copies/mL. Either Pearson's 2 test or Fisher's exact test was applied to evaluate the heterogeneity of contingency tables. Differences between means and distributions were evaluated by the Mann-Whitney U test and the 2-tailed Student's t test. Associations between variables were determined by Pearson's correlation coefficient. Multiple linear regression analyses were conducted to evaluate the associations between the dependent variables FEF25%75%, FEV1/FVC, and FEF25%75%/FVC and several independent variables, by use of SPSS for Windows (version 8.0; SPSS). P < .05 was considered to be statistically significant.
RESULTS
Nasal TTV in the children with asthma and the healthy control children.
The nasal fluids of 55 (93%) of the 59 children with asthma tested positive for TTV loads, which ranged from 3.9 to 7.8 log10 DNA copies/mL of nasal fluid (geometric mean ± SD TTV load, 5.9 ± 1.1 log10 DNA copies/mL of nasal fluid). These values did not differ significantly from those obtained for the nasal fluids of the 30 control children who had no history of asthma (rate of positivity for TTV, 83%; geometric mean ± SD TTV load, 5.5 ± 1.1 log10 DNA copies/mL of nasal fluid).
Lung function and nasal TTV loads in the children with asthma and the healthy control children.
As was expected given their satisfactory clinical conditions, the children with asthma exhibited overall spirometric indices that were similar to those exhibited by the control children. However, as was also expected given their asthmatic states, the mean FEF25%75%, FEV1/FVC, and FEF25%75%/FVC indices for the children with asthma were significantly reduced, compared with those of the control children (table 1). All of the children with asthma and all of the control children were then analyzed for possible associations between lung function and nasal TTV load. Because too few children had no detectable TTV in nasal fluid to permit meaningful comparisons, the children were stratified into 2 categories, those with low (<6.0 log10 DNA copies/mL of nasal fluid) and high (6.0 log10 DNA copies/mL of nasal fluid) nasal TTV loads. By analogy, the cutoff of 6 log10 DNA copies/mL of nasal fluid was used on the basis of previous findings showing that serum ECP levels were significantly increased in young children with acute respiratory diseases who had plasma TTV loads 6 log10 DNA copies/mL [7]. In the children with asthma, comparison of the spirometric indices showed that (1) FEF25%75%values that were <70% of the predicted normal values were more frequent in the children with asthma who had high nasal TTV loads than they were in those who had low TTV viral loads (8/24 vs. 4/35; P = .04) and (2) mean FEF25%75%, FEV1/FVC, and FEF25%75%/FVC values were significantly lower in the children with asthma who had high TTV loads than they were in those who had low TTV loads (table 1). Moreover, a significant inverse correlation was observed between the spirometric indices and nasal TTV load in the children with asthma (figure 1); in contrast, no correlation whatsoever was detected between the spirometric indices and nasal TTV load in the control children (data not shown).
No other significant differences between the children with asthma who had high TTV loads and those who had low TTV loads were observed for a number of anamnestic, clinical, and therapeutic variables, including sensitivity to aeroallergens, as determined by skin-prick tests. Rhinoviruses, the respiratory viruses that are possibly the most frequently associated with asthma attacks [25], were detected in 1 child with asthma who had a low TTV load and in 4 children with asthma who had high TTV loads, but the difference was not statistically significant (table 2). Several variables may influence lung function in children with asthma, including duration of symptomatic disease and characteristics of treatment [12, 13, 26, 27]. As is shown in table 3, when a number of such variables and high nasal TTV load were examined by multiple linear regression analysis for a possible influence on FEF25%75%, FEV1/FVC, and FEF25%75%/FVC, a significant negative correlation was observed for nasal TTV load only. Of the remaining variables, duration of therapy and dose of inhaled corticosteroids showed a positive correlation, and the others showed none at all.
Nasal TTV genogroups in the children with asthma.
TTV is classified into 5 genogroups, designated 15 [28]. It was, therefore, of interest to assess whether the presence of selected TTV genogroups or of multiple genogroups in the respiratory tract of the children with asthma might be associated with diminished spirometric indices. The nasal specimens of each of the 55 TTV-positive children with asthma were characterized by amplification by 5 distinct PCR protocols, each of which was specific for a TTV genogroup. Table 4 shows that this typing protocol identified the TTV genogroup for 49 of the children with asthma. In these children, the distribution of the TTV genogroups and the frequency of multiple-genogroup infections were similar to what was previously observed in the general population of the same geographical area [4, 6, 7], with the possible exception of a slightly higher representation of genogroup 5. Also, the mean FEF25%75%, FEF25%75%/FVC, and FEF1/FVC indices of these children did not differ significantly depending on the number or identity of the TTV genogroups carried. In 6 TTV-positive children with asthma, the virus carried could not be amplified by any of the genogroup-specific PCR assays; given that the nasal TTV loads of these children were particularly low (geometric mean ± SD, 4.9 ± 0.4 log10 DNA copies/mL of nasal fluid), this was most likely due, at least in part, to the difference in sensitivity between the PCR assay used for TTV detection and the PCR assays used for typing (lower limits of detection, 1000 and 4000 DNA copies/mL of nasal fluid, respectively). Interestingly, these 6 children also had higher mean spirometric indices than did the rest of the children with asthma (table 4).
Sputum ECP levels and cellularity in the children with asthma, by nasal TTV load.
Table 5 shows the mean cellularity and ECP levels in the sputum of those children with asthma who yielded valid specimens, stratified by whether the children had high or low nasal TTV loads. Sputum cellularity was similar in the 2 groups of children. However, the mean ECP level was significantly increased in the children with asthma who had high TTV loads, compared with that in the children with asthma who had low TTV loads. Furthermore, ECP level and nasal TTV load were correlated (r = 0.357; P = .045), and ECP level and cellularity were unrelated to spirometric indices.
DISCUSSION
The connections between viral infections and childhood asthma have been the subject of much investigation and debate. It is now generally accepted that acute viral respiratory tract infections can precipitate asthma attacks by increasing airway responses to nonspecific environmental stimuli as well as by other means [2931]. Furthermore, respiratory syncytial virus bronchiolitis early during life has been associated with an increased risk of future recurrent wheezing illnesses and asthma [32, 33]. Finally, certain persisting virusesadenoviruses, for examplehave been implicated as possibly playing a role in the facilitation or aggravation of allergen-induced lung inflammation [34] and in the development of steroid resistance in asthma [35]. However, this and other empirical evidence cannot be considered conclusive. The childhood of nearly everyone is punctuated by a generally vast number of acute respiratory viral infections, and yet only a minority go on to develop wheezing illnesses or asthma; this suggests that these most likely result from the interplay of multiple concurrent factors, with viruses being one of the possible players [31, 36, 37].
Although TTV infection is quite common and highly persistent [3], the present study is, to our knowledge, the first assessment of the status of TTV infection in children with asthma. Ninety-three percent of the 59 children with asthma and 83 percent of the 30 matched healthy control children were found to harbor TTV in nasal fluid. The results also showed that, among individual children, nasal TTV load (an indicator of the replicative activity of the virus in the upper respiratory tract) varied extensivelyspecifically, over a range of 4 log10 DNA copies/mL of nasal fluid. Such high rates of TTV positivity and such a wide viral load range were expected, in light of previous findings in younger children with acute respiratory diseases [4, 6, 7] and in the general population [20] of the same geographical area.
We then evaluated whether nasal TTV load was related to respiratory conditions. Because meaningful comparisons between TTV-positive and TTV-negative children were prevented by the low number of the latter, we compared the children with high nasal TTV loads (i.e., viral loads 6.0 log10 DNA copies/mL of nasal fluid) and the children with low or undetectable nasal TTV loads. Interestingly, in the children with asthma but not in the control children, high TTV nasal loads were associated with decrements in all of the spirometric indices measured, although statistical significance was reached only for FEF25%75%, FEF1/FVC, and FEF25%75%/FVC. These have been shown to be sensitive spirometric indicators of subtle airway dysfunction and are considered to be markers of the levels of medium and small airway obstruction [38, 39]. The size of the mean decrements observed were relatively modest, but it appears to be likely that the differences would have been larger if the same comparisons could have been made between TTV-positive and TTV-negative children. Indeed, it may not be by chance that the 4 children with asthma who tested negative for TTV exhibited particularly good spirometric indices (data not shown). It is also important to note that the predicted normal spirometric indices with which those of the study children were compared had been established in subjects whose TTV infection status was not known but who, it can be presumed on the basis of what has emerged recently about the high prevalence of TTV, were mostly TTV infected. Unfortunately, the pervasiveness of TTV infection will make it very laborious to examine the lung functions of individuals without detectable TTV, but the present results indicate that efforts in this direction would be worthwhile.
Being highly heterogeneous genetically, TTV is currently classified into 5 widely divergent genogroups [28]. In an attempt to assess whether identity and/or variety of the infecting TTV could bear on airway function, we determined the genogroup(s) of the TTV present in the nasal fluids of all of the TTV-positive children with asthma. The results, however, revealed no evidence that the TTV genogroups present in the children with asthma differed from those in the control children or of a preferential association between either a specific TTV genogroup or multiple TTV genogroups and low spirometric indices. This suggests that it is the overall extent of TTV replication, and not the characteristics and variety of the TTV replicating, in the respiratory tract that may impinge on airway function.
The reasons for the interesting association between sustained TTV replication in the upper respiratory tract and inferior airway function that was observed in the children with asthma but not in the control children remain to be elucidated. Nasal TTV load was unrelated to all therapy variables examined, thus arguing against the possibility that it was dependent on variations in treatment. It is, therefore, plausible that TTV negatively impacts airway size and/or tone in children with asthma. For example, TTV might produce fine airway alterations either directly or, as the elevated sputum ECP levels detected in the children with asthma who had high nasal TTV loads might point to, through the inflammatory response elicited. In addition, similar to what has been seen in children with acute respiratory diseases [6], florid TTV replication might help to skew the systemic or local immune system toward Th2 responses, which are believed to be critical to the pathogenesis of asthma [36]. Unlike what is observed for many other infectious agents, TTV infection is extremely common even under high-sanitation conditions [3]. Continued and sustained respiratory contact with TTV (and possibly with the other anellovirus, torquetenominivirus [19]), coupled with reduced exposure to other infectious and environmental stimuli that tend to orientate toward a predominance of Th1 responses, might be a factor in the increased prevalence of asthma noted in developed countries during recent decades [40]. However, because other inflammatory conditions have been associated with high TTV loads [4, 41], the present data are also compatible with the possibility that enhanced TTV replication merely identifies children with inherently reduced airway function or results from other pathophysiological changes that occur in asthma, such as an augmented cycling of local lymphoid cells [42] or inflammation itself. Further research in the area is clearly warranted.
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