Virologia.unipi.it

Associations between Nasal Torquetenovirus Loadand Spirometric Indices in Children with Asthma Massimo Pifferi,1 Fabrizio Maggi,2 Elisabetta Andreoli,2 Letizia Lanini,2 Emanuela De Marco,1 Claudia Fornai,2
Maria Linda Vatteroni,2 Mauro Pistello,2 Vincenzo Ragazzo,1 Pierantonio Macchia,1 Attilio Boner,3
and Mauro Bendinelli2
1Department of Pediatrics and 2Virology Section and Retrovirus Center, Department of Experimental Pathology, University of Pisa, Pisa,and 3Department of Pediatrics, University of Verona, Verona, Italy (See the editorial commentary by Piedra and the brief report by Williams et al., on pages 1138–40 and 1149–53,
respectively.)
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 function—forced expiratory flow (FEF) in which 25% and 75% of forced vital
capacity (FVC) is expired (FEF
), forced expiratory volume in 1 s/FVC, and FEF
/FVC—showed an
25%–75%
25%–75%
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 (у6 log DNA copies/mL of nasal fluid) than they were
in those who had low nasal TTV loads (!6 log
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 Anel- certainties about what possible pathogenic potential it lovirus, which was recently established (but has not yet been classified within any viral family) to accommodate Recently, by studying children р2 years old who had a number of small viruses with single-stranded circular acute respiratory diseases, we and others demonstrated DNA genomes that could not be classified within the that the respiratory tract is a site of primary infection family Circoviridae [1]. Early studies after its discov- and continual TTV replication [4, 5]. Although no evi- ery in 1997 focused on TTV as the possible causative dence was obtained to suggest that TTV is a direct cause agent of cryptogenetic hepatitis, but the observation of respiratory disease, mean TTV loads in plasma and that chronic TTV viremia is widespread among the gen- nasal fluids were considerably higher in children with eral population worldwide has led to considerable un- bronchopneumonia than they were in those with milderillnesses [4]. Furthermore, TTV loads were negativelyrelated to the percentages of circulating CD3+ and CD4+ Received 29 November 2004; accepted 17 February 2005; electronically T cells and were positively related to the percentage of circulating B cells, suggesting that TTV might have im- Potential conflicts of interest: none reported.
Financial support: Ministero dell’Istruzione, dell’Universita` e della Ricerca, Rome, munomodulatory effects [6]. Finally, the presence and load of TTV in plasma were found to correlate with Reprints or correspondence: Prof. Mauro Bendinelli, Virology Section and Retrovirus Center, Dept. of Experimental Pathology, University of Pisa, via San the levels of eosinophil cationic protein (ECP) in se- Zeno 37, I-56127 Pisa, Italy (bendinelli@biomed.unipi.it).
rum [7]. Because it has been proposed that high serum The Journal of Infectious Diseases
2005; 192:1141–8
ECP levels in young children predict an increased likeli- ᮊ 2005 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2005/19207-0003$15.00 hood of developing airway hyperreactivity, wheezing ill- • JID 2005:192 (1 October) • 1141
nesses, and asthma at later times [8–11], we hypothesized that in the maximum inspiratory position, until at least 2 compa- TTV infection might represent a heretofore unrecognized in- rable flow-volume curves were obtained (i.e., with FEV and ducer or, more likely, facilitator in the pathogenesis of these FVC values differing by no more than 5%) [15]. The results are expressed as a percentage of the normal values predicted Prompted by the above findings, in the present study we as- for children of the same age, height, and body surface area, sessed the status of TTV infection in the upper respiratory tract of a well-characterized group of children with well-controlled, Specimen collection and processing.
mild to moderate persistent asthma and examined whether TTV lected and rapidly transferred to the clinical virology laboratory infection status was related to lung function. Our results suggest of the Department of Experimental Pathology, University of that TTV might indeed contribute to the pathogenesis of asthma.
Pisa, where they were immediately processed, as described else-where [4]. Briefly, the nasal-secretion volume was measured, PARTICIPANTS, MATERIALS, AND METHODS
and then secretions were diluted in PBS and centrifuged. Cell-free nasal fluid was aspirated and stored in aliquots at Ϫ80ЊC Study populations.
until use. For sputum induction, the children were premedi- children with well-controlled, mild to moderate persistent asthma cated with 200 mg of inhaled salbutamol, to inhibit possible (51 boys and 8 girls; mean ע SD age, 11.5 ע 2.2 years; age bronchoconstriction, and were then exposed via inhalation to range, 7–16 years) admitted for a medical examination to the a 3.5% NaCl aerosol solution generated by an ultrasonic neb- Department of Pediatrics, University Hospital of Pisa, from Jan- ulizer (Omron U1; Omron Healthcare) for a total of 20 min.
uary 2002 to July 2003. Asthma was diagnosed in accordance FEV was recorded before and 10 min after salbutamol ad- with the recommendations of the Global Initiative for Asthma ministration and then every 5 min during saline inhalation.
[12, 13]. All children were born after at least 37 weeks of gestation, The nebulization was stopped when FEV decreased by у20% weighed 12700 g at birth (mean weight, 3547 ע 422 g; weight from baseline value or when symptoms of bronchoconstric- range, 2770–4250 g), were nonsmokers, and were serologically tion occurred [17]. The children were instructed to rinse their negative for hepatitis B virus surface antigen and antibodies to mouths, to minimize salivary contamination, and were en- hepatitis C virus and HIV. None had a history of receiving an- couraged to cough deeply into a sterile plastic container. Valid tiviral drugs, blood, or blood products. At the time of testing, sputum specimens were, however, produced by only 32 chil- all of the children were asymptomatic, having had no signs of dren. These were processed within 2 h of collection by a method acute asthma or exacerbations for at least 2 months, and were that has been described elsewhere [18]. Briefly, after treatment receiving long-term treatment with inhaled corticosteroids at with 0.1% dithiotreithol and complete homogenization, the doses ranging from 200 to 1000 mg/day. Twenty-one of the chil-dren with asthma were also receiving long-acting b -agonists (19 sputum specimens were filtered to remove cell debris and mu- salmeterol and 2 formoterol), and 15 were also receiving mon- cus, and the total number of nonsquamous cells was counted telukast. Thirty age-matched healthy children served as the con- manually by use of a hemocytometer (Heinz-Herenz). After trol subjects; these children lived in the same area as the ones centrifugation, the sputum supernatants were stored at Ϫ70ЊC with asthma but had a negative history of asthma, atopy, and for later ECP analysis, and resuspended cell pellets were used wheezing, as determined by questions that were based on rec- for cytocentrifuge slide preparation. Differential cell counts ommended questionnaires [11]. All enrolled participants were were performed on a minimum of 500 nonsquamous cells and physically examined and were assessed for lung function by spi- are expressed as percentages of these cells. Sputum ECP levels rometry. On the same day, nasal fluids were collected and stored were determined by a commercial radioimmunoassay (UniCAP until being tested in a blinded fashion for the presence and load 100; Pharmacia and Upjohn), in accordance with the manu- of TTV. The children with asthma also received concomitant facturer’s instructions. All sputum ECP levels were measured skin-prick tests with a panel of standardized allergen extracts [14] in duplicate; the intra- and interassay coefficients of variation and provided sputum specimens. Informed consent was obtained were !3%, and the lower limit of detection was 2 ng/mL.
from the parents of all children who provided specimens.
TTV detection and quantification.
Lung function tests.
tracted from 200 mL of nasal fluid by use of the QIAamp DNA expiratory flow (FEF) volume maneuvers were performed by Mini Kit (QIAgen). Presence and load of TTV were determined use of MasterScreen Body equipment (Jaeger). Supervised by in triplicate by a universal TaqMan real-time polymerase chain a single experienced examiner (M.P.), measurements were taken reaction (PCR) assay targeted to a highly conserved segment with the child seated and included forced expiratory volume of the noncoding region of the viral genome. This assay is po- in 1 s (FEV ), forced vital capacity (FVC), and FEF where 25% tentially capable of amplifying all of the genetic forms of TTV hitherto recognized at a lower limit of detection of 1000 DNA formed a minimum of 3 forced expiratory maneuvers, starting copies/mL of nasal fluid [4, 6, 7] but, as determined by sequence 1142 • JID 2005:192 (1 October) • Pifferi et al.
Lung function in the children with asthma, by nasal torquetenovirus (TTV) load, and
in the matched healthy control children.
Data are mean ע SD percentages of predicted normal values. FEF which 25% and 75% of FVC is expired; FEV , forced expiratory volume in 1 s; FVC, forced vital capacity.
a The cutoff for high and low nasal TTV loads was considered to be 6 log on the basis of previous findings [7].
However, the statistical differences were conserved when !5.0 log was considered to be a low viral load and у6.0 log was considered to be a high viral load.
b Statistically different, compared with the index for the control children (P p c Statistically different, compared with the index for the children with asthma who had nasal TTV loads !6.0 log10 d Statistically different, compared with the index for the control children (P p e Statistically different, compared with the index for the children with asthma who had nasal TTV loads !6.0 log10 f Statistically different, compared with the index for the control children (P p g Statistically different, compared with the index for the children with asthma who had nasal TTV loads !6.0 log10 data, does not amplify the other anellovirus, which is known Either Pearson’s x2 test or Fisher’s exact test was applied to as “torquetenominivirus” because of its smaller genome [19].
evaluate the heterogeneity of contingency tables. Differences The procedures used for the quantification of copy numbers between means and distributions were evaluated by the Mann- and the evaluation of specificity, sensitivity, intra- and interassay Whitney U test and the 2-tailed Student’s t test. Associations precision, and reproducibility of the assay have been described between variables were determined by Pearson’s correlation coefficient. Multiple linear regression analyses were conducted TTV characterization.
to evaluate the associations between the dependent variables positive by the universal PCR assay were amplified by 5 distinct PCR protocols, each of which was specific for a TTV genogroup pendent variables, by use of SPSS for Windows (version 8.0; and had a lower limit of detection of ∼4000 DNA copies/mL SPSS). P ! .05 was considered to be statistically significant.
of nasal fluid. The specificities and sensitivities of these assays,as well as the modification of certain primer sequences intro- duced to expand the breadth of detection, have been describedelsewhere [4, 6, 7, 21]. All specimens were tested at least in Nasal TTV in the children with asthma and the healthy control
children.
The nasal fluids of 55 (93%) of the 59 children with Rhinovirus detection.
asthma tested positive for TTV loads, which ranged from 3.9 to were free from exacerbations at the time of testing, they were 7.8 log DNA copies/mL of nasal fluid (geometric mean ע SD not systematically examined for other common pathogens that TTV load, 5.9 ע 1.1 log DNA copies/mL of nasal fluid). These have been implicated in the pathogenesis of exacerbations [22, values did not differ significantly from those obtained for the 23]. However, all of the children were screened for nasal carriage nasal fluids of the 30 control children who had no history of of rhinoviruses, to assess for a possible association with TTV asthma (rate of positivity for TTV, 83%; geometric mean ע SD presence and load. The method used consisted of an in-house TTV load, 5.5 ע 1.1 log DNA copies/mL of nasal fluid).
nested reverse-transcriptase PCR assay targeted to the con- Lung function and nasal TTV loads in the children with
served noncoding region of the viral genome [24].
asthma and the healthy control children.
Statistical analyses.
ed given their satisfactory clinical conditions, the children with performed on log -transformed values, to approximate a nor- asthma exhibited overall spirometric indices that were similar to mal distribution, and with the children without detectable TTV those exhibited by the control children. However, as was also considered to have a viral load of 3.0 log expected given their asthmatic states, the mean FEF TTV in Children with Asthma • JID 2005:192 (1 October) • 1143
were in those who had low TTV viral loads (8/24 vs. 4/35;P p FVC values were significantly lower in the children with asthmawho had high TTV loads than they were in those who had lowTTV loads (table 1). Moreover, a significant inverse correlationwas observed between the spirometric indices and nasal TTVload in the children with asthma (figure 1); in contrast, no cor-relation whatsoever was detected between the spirometric indicesand 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 TTVloads were observed for a number of anamnestic, clinical, andtherapeutic variables, including sensitivity to aeroallergens, asdetermined by skin-prick tests. Rhinoviruses, the respiratoryviruses that are possibly the most frequently associated withasthma attacks [25], were detected in 1 child with asthma whohad a low TTV load and in 4 children with asthma who hadhigh TTV loads, but the difference was not statistically signif-icant (table 2). Several variables may influence lung functionin children with asthma, including duration of symptomaticdisease and characteristics of treatment [12, 13, 26, 27]. As isshown in table 3, when a number of such variables and highnasal TTV load were examined by multiple linear regressionanalysis for a possible influence on FEF /FVC, a significant negative correlation was observed for nasal TTV load only. Of the remaining variables, durationof therapy and dose of inhaled corticosteroids showed a positivecorrelation, and the others showed none at all.
Figure 1.
Correlations between nasal torquetenovirus (TTV) load and Nasal TTV genogroups in the children with asthma.
predicted normal values for forced expiratory flow (FEF) in which 25% and is classified into 5 genogroups, designated 1–5 [28]. It was, 75% of forced vital capacity (FVC) is expired (FEF therefore, of interest to assess whether the presence of selected TTV genogroups or of multiple genogroups in the respiratorytract of the children with asthma might be associated with /FVC indices for the children with asthma diminished spirometric indices. The nasal specimens of each were significantly reduced, compared with those of the control of the 55 TTV-positive children with asthma were characterized children (table 1). All of the children with asthma and all of the by amplification by 5 distinct PCR protocols, each of which control children were then analyzed for possible associations be- was specific for a TTV genogroup. Table 4 shows that this typing tween lung function and nasal TTV load. Because too few chil- protocol identified the TTV genogroup for 49 of the children dren had no detectable TTV in nasal fluid to permit meaningful with asthma. In these children, the distribution of the TTV comparisons, the children were stratified into 2 categories, those genogroups and the frequency of multiple-genogroup infec- with low (!6.0 log DNA copies/mL of nasal fluid) and high tions were similar to what was previously observed in the gen- (у6.0 log DNA copies/mL of nasal fluid) nasal TTV loads. By eral population of the same geographical area [4, 6, 7], with analogy, the cutoff of 6 log DNA copies/mL of nasal fluid was the possible exception of a slightly higher representation of used on the basis of previous findings showing that serum ECP levels were significantly increased in young children with acute FEF /FVC indices of these children did not differ significantly respiratory diseases who had plasma TTV loads у6 log DNA depending on the number or identity of the TTV genogroups copies/mL [7]. In the children with asthma, comparison of the carried. In 6 TTV-positive children with asthma, the virus car- ried could not be amplified by any of the genogroup-specific !70% of the predicted normal values were more frequent in the PCR assays; given that the nasal TTV loads of these children children with asthma who had high nasal TTV loads than they were particularly low (geometric mean ע SD, 4.9 ע 0.4 log10 1144 • JID 2005:192 (1 October) • Pifferi et al.
Characteristics of the children with asthma, by nasal torquetenovirus (TTV) load.
Duration since first symptoms, mean ע SD, years Inhaled corticosteroids, mean ע SD, mg/day Duration of therapy with corticosteroids, mean ע SD, years Data are no. of children, unless otherwise noted. MiP, mild persistent; MoP, moderate persistent; a Determined on the basis of clinical symptoms.
DNA copies/mL of nasal fluid), this was most likely due, at Sputum ECP levels and cellularity in the children with
least in part, to the difference in sensitivity between the PCR asthma, by nasal TTV load.
assay used for TTV detection and the PCR assays used for larity and ECP levels in the sputum of those children with typing (lower limits of detection, 1000 and ∼4000 DNA copies/ asthma who yielded valid specimens, stratified by whether the mL of nasal fluid, respectively). Interestingly, these 6 children children had high or low nasal TTV loads. Sputum cellular- also had higher mean spirometric indices than did the rest of ity was similar in the 2 groups of children. However, the mean ECP level was significantly increased in the children with Multiple linear regression analysis on the dependent variables forced expiratory flow (FEF) in which
25% and 75% of forced vital capacity (FVC) is expired (FEF
), forced expiratory volume in 1 s (FEV )/FVC, and
25%–75%
/FVC and various independent variables for the children with asthma.
25%–75%
Nasal TTV load у6 log DNA copies/mL of nasal fluid Duration of therapy with inhaled corticosteroids Positive skin-prick test for dust mitesa,b NS, not significant; TTV, torquetenovirus.
a Variable removed from the last model with the dependent variables FEF b Variable removed from the last model with the dependent variable FEV /FVC because its P value was not statistically significant TTV in Children with Asthma • JID 2005:192 (1 October) • 1145
Lung function and torquetenovirus (TTV) genogroups detected in nasal fluid from the
children with asthma.
, forced expiratory flow in which 25% and 75% of FVC is expired; FEV , forced expiratory volume a Geometric mean ע SD log DNA copies/mL of nasal fluid.
b Data are mean ע SD percentages of predicted normal values.
c Specimens that were positive for TTV by a universal polymerase chain reaction (PCR) assay but were negative by all 5 genogroup-specific PCR assays.
d Statistically different, compared with loads for the 49 children who were positive by at least 1 genogroup-specific e Statistically different, compared with the indices for the 49 children who were positive by at least 1 genogroup- f Statistically different, compared with the indices for the 49 children who were positive by at least 1 genogroup- g Statistically different, compared with the indices for the 49 children who were positive by at least 1 genogroup- asthma who had high TTV loads, compared with that in the Finally, certain persisting viruses—adenoviruses, for example— children with asthma who had low TTV loads. Furthermore, have been implicated as possibly playing a role in the facilitation ECP level and nasal TTV load were correlated (r p 0.
or aggravation of allergen-induced lung inflammation [34] and .045 and ECP level and cellularity were unrelated to spi- in the development of steroid resistance in asthma [35]. How- ever, this and other empirical evidence cannot be consideredconclusive. The childhood of nearly everyone is punctuated by DISCUSSION
a generally vast number of acute respiratory viral infections, The connections between viral infections and childhood asthma and yet only a minority go on to develop wheezing illnesses or have been the subject of much investigation and debate. It is asthma; this suggests that these most likely result from the now generally accepted that acute viral respiratory tract infec- interplay of multiple concurrent factors, with viruses being one tions can precipitate asthma attacks by increasing airway re- of the possible players [31, 36, 37].
sponses to nonspecific environmental stimuli as well as by other Although TTV infection is quite common and highly per- means [29–31]. Furthermore, respiratory syncytial virus bron- sistent [3], the present study is, to our knowledge, the first as- chiolitis early during life has been associated with an increased sessment of the status of TTV infection in children with asthma.
risk of future recurrent wheezing illnesses and asthma [32, 33].
Ninety-three percent of the 59 children with asthma and 83 Cellularity and eosinophil cationic protein (ECP) levels in sputum from the children with asthma, by nasal torquetenovirus
(TTV) load.
Eosinophils, Lymphocytes, Monocytes, Neutrophils, a A sputum specimen for cell-type counts was obtained from only 22 subjects.
b Statistically different, compared with the ECP level for the children with asthma who had nasal TTV loads !6.0 log (P p 1146 • JID 2005:192 (1 October) • Pifferi et al.
percent of the 30 matched healthy control children were found variety of the TTV replicating, in the respiratory tract that may to harbor TTV in nasal fluid. The results also showed that, among individual children, nasal TTV load (an indicator of the The reasons for the interesting association between sustained replicative activity of the virus in the upper respiratory tract) TTV replication in the upper respiratory tract and inferior air- varied extensively—specifically, over a range of 4 log way function that was observed in the children with asthma copies/mL of nasal fluid. Such high rates of TTV positivity and but not in the control children remain to be elucidated. Nasal such a wide viral load range were expected, in light of previous TTV load was unrelated to all therapy variables examined, thus findings in younger children with acute respiratory diseases [4, arguing against the possibility that it was dependent on vari- 6, 7] and in the general population [20] of the same geograph- ations in treatment. It is, therefore, plausible that TTV nega- tively impacts airway size and/or tone in children with asthma.
We then evaluated whether nasal TTV load was related to For example, TTV might produce fine airway alterations either respiratory conditions. Because meaningful comparisons between directly or, as the elevated sputum ECP levels detected in thechildren with asthma who had high nasal TTV loads might TTV-positive and TTV-negative children were prevented by the point to, through the inflammatory response elicited. In ad- low number of the latter, we compared the children with high dition, similar to what has been seen in children with acute nasal TTV loads (i.e., viral loads у6.0 log DNA copies/mL of respiratory diseases [6], florid TTV replication might help to nasal fluid) and the children with low or undetectable nasal TTV skew the systemic or local immune system toward Th2 re- loads. Interestingly, in the children with asthma but not in the sponses, which are believed to be critical to the pathogenesis control children, high TTV nasal loads were associated with dec- of asthma [36]. Unlike what is observed for many other in- rements in all of the spirometric indices measured, although fectious agents, TTV infection is extremely common even under statistical significance was reached only for FEF high-sanitation conditions [3]. Continued and sustained re- /FVC. These have been shown to be sensitive spi- spiratory contact with TTV (and possibly with the other anel- rometric indicators of subtle airway dysfunction and are consid- lovirus, torquetenominivirus [19]), coupled with reduced ex- ered to be markers of the levels of medium and small airway posure to other infectious and environmental stimuli that tend obstruction [38, 39]. The size of the mean decrements observed to orientate toward a predominance of Th1 responses, might were relatively modest, but it appears to be likely that the dif- be a factor in the increased prevalence of asthma noted in ferences would have been larger if the same comparisons could developed countries during recent decades [40]. However, be- have been made between TTV-positive and TTV-negative chil- cause other inflammatory conditions have been associated with dren. Indeed, it may not be by chance that the 4 children with high TTV loads [4, 41], the present data are also compatible asthma who tested negative for TTV exhibited particularly good with the possibility that enhanced TTV replication merely iden- spirometric indices (data not shown). It is also important to note tifies children with inherently reduced airway function or re- that the predicted normal spirometric indices with which those sults from other pathophysiological changes that occur in of the study children were compared had been established in asthma, such as an augmented cycling of local lymphoid cells subjects whose TTV infection status was not known but who, it [42] or inflammation itself. Further research in the area is can be presumed on the basis of what has emerged recently about the high prevalence of TTV, were mostly TTV infected. Unfor-tunately, the pervasiveness of TTV infection will make it verylaborious to examine the lung functions of individuals without References
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