This prospective study was carried out at the Department of Maternal and Pediatric Sciences of the University of Milan, Italy, during the winter seasons 2007-2008 and 2008-2009. The protocol was approved by the local Ethics Committee, and written informed consent to study participation was obtained from the patients' parents or legal guardians.
The study involved otherwise healthy infants aged 1-12 months who were admitted to hospital because of bronchiolitis during the study period. The exclusion criteria were the presence of a chronic disease increasing the risk of complications of respiratory infection, including chronic disorders of the pulmonary or cardiovascular system, chronic metabolic disease, neoplasms, kidney or liver dysfunction, hemoglobinopathies, immunosuppression, and genetic or neurological disorders. There was no refusal to participate.
Upon admission, the infants' demographic characteristics and medical history were systematically recorded using standardised written questionnaires and, after a complete physical examination, the subjects with a diagnosis of bronchiolitis based on well-established criteria  were enrolled. The severity of the disease was defined on the basis of a global evaluation of the signs and symptoms. In particular, on the basis of previously published criteria , respiratory illness was considered severe in the presence of all of ≤ 92% pulse oximetry, a respiratory rate of ≥60 breaths/min, marked accessory muscle use, nasal flare or grunting, a heart rate of > 180 beats/min, an inability to feed and a toxic appearance. All of the patients underwent chest radiography, and pneumonia was defined on the basis of the presence of a reticular-nodular infiltrate, segmental or lobar consolidation, or bilateral consolidation .
Upon enrolment, Virocult (Medical Wire and Equipment, Corsham, UK) nasopharyngeal swabs were used to collect specimens for the detection of RSV, and a blood sample was drawn to assess troponin I concentrations. On the basis of our previous experience in children with bronchiolitis in which we showed that RSV was the main cause of acute episodes in hospitalized children , in this study only RSV was searched on nasopharyngeal secretions. Finally, on the day of admission, all of the infants underwent 24-hour Holter ECG monitoring and a complete heart evaluation with echocardiography. It was decided to estimate pulmonary pressure as well as signs of pulmonary hypertension only in presence of pathologic findings at echocardiography.
During their hospital stay, the infants' clinical signs and symptoms were monitored daily. They were treated with oxygen when saturation was ≤ 95%, and received inhalatory bronchodilators, steroids, antibiotics, intravenous fluids and chest physiotherapy on the basis of the judgement of the attending pediatrician. They were discharged when they were able to maintain > 95% oxymetry without oxygen, but their parents were asked to bring them immediately to the study centre if there were any recurrent or worsening signs and symptoms.
The medical history, general physical condition and clinical symptoms of each patient were re-evaluated by investigators blinded to the etiological and cardiac findings four weeks after enrolment. During this follow-up visit, the patients' history of respiratory tract infections was carefully assessed and 24-hour Holter ECG monitoring 24 hours was repeated.
Identification of RSV virus
The Virocult nasopharyngeal swabs were tested by means of previously described real-time polymerase chain reaction (PCR) for RSV types A and B [21–24], with total nucleic acids being routinely isolated at the MagnaPureLC Isolation Station (Roche Applied Science, Penzberg, Germany). A universal internal control virus (phocine distemper virus, PDV) was used to monitor the whole process from nucleic acid isolation to real-time detection. The in-house real-time PCRs for RSV and PDV were designed using primer express software (Applied Biosystem, Nieuwerkerk a/d Ijssel, The Netherlands).
RNA was amplified in a single tube, two-step reaction using Taqman reverse transcriptase and PCR core reagent kits (Applied Biosystems, Foster City, CA, USA) and an ABI 7700 or ABI 7500 sequence detection system (Applied Biosystems). A cultured positive control virus was used for each assay. On the basis of proficiency testing data, the sensitivity of each assay was estimated to be less than 500 copies ⁄ mL.
Evaluation of myocardial damage
To evaluate myocardial damage, serum troponin I levels were measured using the Abbott AxSYM system (Abbott Laboratories, Mississauga, Ontario, Canada) at the time of hospital admission, and were considered indicative of myocardial damage when they were > 1.2 μg/L. The measurements had a coefficient of variation of 10%, and the lower detection limit was 0.3 μg/L.
Holter ECG monitoring
Three-channel Holter monitors (ElaMedical Spider View 3 channel recorders, Le Plessis-Robinson, France) were positioned immediately after hospital admission, and 24-hour recordings were obtained. After the skin had been prepared, the electrodes were placed to record leads II, V1 and V5; a 1 mV calibration signal was also recorded. The built-in clock started after the electrodes had been attached.
A commercial Holter analysis software (SyneScope, Elamedical, Sorin Group, Le Plessis-Robinson, France) was used to analyse rhythm and heart rate variability (HRV, time-, frequency- and geometric-domain indices) from the Holter tapes. QRS was detected using a level detector, but was manually over-read by a physician. All of the tapes were edited in order to assure the accuracy of the QRS classification. Ectopic beats, noisy data, and artifacts were manually identified and excluded from the HRV analysis. Non-stationarities were avoided by means of trigger adjustment. Average hourly heart rates were determined from the computerised Holter scanner, and maximum, minimum, and mean 24-hour heart rates (with standard deviations, SDs) were calculated for each subject.
The time-domain parameters measured from the Holter tapes were: 1) the average of all normal-to-normal beats (mean NN interval) (mean heart rate); 2) the SD of all NN intervals (SDNN); 3) the SD of the average of NN intervals in all 5-minute segments of the 24-hour recording (SDANN); 4) the mean of the standard deviation in all 5-minute segments of the 24-hour recording (ASDNN); 5) the square root of the mean of the squares of the differences between adjacent NN intervals (rMSSD); and 6) the percentage of > 50 msec differences between adjacent NN intervals. Frequency-domain heart rate variability was also determined, including low-frequency power (LF, total NN interval spectral power between 0.04 and 0.15 Hz), high-frequency power (HF, total interval spectral power between 0.15 and 0.4 Hz), and the LF/HF ratio .
The echocardiographic studies were made using a real-time ultrasound imaging system system (Acuson Sequoia 512) equipped with 3-, 5-, 7 and 10A, MHz transducers. The echocardiographic measurements were made using standard techniques .
M-mode measurements were made in accordance with the recommendations of the Cornmittee of M-Mode Standardization of the American Society of Echocardiography , and were used to determine right ventricular internal dimension in diastole (RVID) and left ventricular internal dimensions in diastole (LVID) and systole (LVIS). Left ventricular function was assessed by calculating the percentage fractional shortening of the internal dimension and ejection fraction using standard formulas. Left ventricular mass was also calculated.
The flow velocities across the mitral, tricuspid, aortic and pulmonary valves were recorded from standard pericordial and subcostal positions using pulsed-wave and continuous-wave Doppler transducers.
Continuous variables are given as mean values ± SD, and categorical variables as numbers and percentages. For the comparison between groups (i.e., RSV-positive vs RSV-negative), the continuous data were analysed using a two-sided Student's test if they were normally distributed (on the basis of the Shapiro-Wilk statistic) or a two-sided Wilcoxon rank-sum test if they were not. For the comparison within group (i.e., admission vs 28 ± 3 days after admission in the RSV-positive and RSV-negative groups, separately), the continuous data were analysed using a paired two-sided Student's test or signed-rank test, as appropriate. Categorical data were analysed using contingency table analysis and the chi-square or Fisher's exact test, as appropriate.