Statistically significant possible related risk factors for viral infections in multivariate analysis were decreasing age upon study entry, increasing weight, and lower educational level of the father (Table 4). The frequency of RSV decreased with increasing age upon study entry and gestational age at birth (Table 5), as we can observe that nearly half of all children hospitalized with RSV LRTI (49%, n = 127) are in the less than 6 months age group. Proportionally, RSV positivity was higher among children born with gestational age <35 weeks (50%, n = 29) compared to children born with a gestational age of >35 weeks (39%, n = 175). For RSV infection, statistically significant risk factors in multivariate analysis were decreasing age upon study entry, decreasing gestational age, attendance of daycare or other collective environment, and lower educational level of the father (Table 4). No statistically significant risk factors were found for other viruses in multivariate analysis, except for increasing age upon study entry in the case of influenza and the following factors in the case of metapneumovirus: increasing gestational age, the presence of bronchopulmonary dysplasia, higher educational level of the mother, and lower educational level of the father (data not shown).

Table 2 displays the numbers of cases of LRTI per centers and overall during the 1-year period of interest. Overall, the relative contribution of cases of LRTI over the whole study period was above 10.0% for the months of April to June 2012 and in March 2013, but there was wider temporal variation when each center was analyzed separately. The overall frequency of any proven viral infection is shown in Table 3. Nearly two-thirds of patients had at least 1 proven viral infection. Overall, 204 subjects tested positive for RSV, for a relative frequency of 40.2% (95% CI, 35.9%–44.7%). RSV A was nearly 3 times as frequent than RSV B. The overall prevalence of each of the other viruses was 17% for rhinovirus, 5.9% for metapneumovirus, 5.5% for parainfluenza and bocavirus, 3.7% for adenovirus, and 3.2% for influenza. Of note, there were no cases of coronavirus 229E. Any codetection (not including subtypes of the same virus) was found in 13.8% of patients. The 2 most frequent codetections were RSV and rhinovirus (3.7% of tested samples), as well as RSV and bocavirus (2.0%). Of note, 9 children had codetection with RSV A and RSV B.

Morbidity due to IBV infection can reach up to 100%. Mortality rate may range from 25 to 30% in young chicks but may increase to 80% as a result of factors that are host-associated (age, immune status), virus-associated (strain, pathogenicity, virulence, and tissue tropism), or environmental (cold and heat stresses, dust, and presence of ammonia). Secondary bacterial infections (E. coli) or coinfection with immunosuppressive viruses such as Marek's disease virus, infectious bursal disease virus (IBDV) [33, 47, 48], may worsen the outcomes of IBV infection. Generally, nephropathogenic IBV strain causes high mortality, compared with strains infecting only the respiratory or reproductive systems.

The virus detection rates for different seasons are shown in Fig. 2. The proportion of positive viruses exhibited two waves corresponding to winter and spring, including Jan to Mar 2015, and Nov 2015 to Feb 2016. Similarly, the FluA virus also occurred more frequently in winter and spring. Conversely, EV infections were predominant between April and September. Other viruses occurred almost sporadically throughout the year without obvious seasonal trends, and a small number of ARI outpatients with virus infection were observed between June and September, excluding EV infections.

The virus has been shown to have a worldwide distribution and was observed primarily in the winter season in temperate climates. On the other hand, countries with extreme weather, like Canada, have also shown virus activity around January to March, although milder symptoms were reported. Interestingly, seasonal variations have been reported in China where infection with HCoV-NL63 appeared mainly in spring and summer. Also, a recent study of coronaviruses in Thailand did not show any seasonal predilection, while Wu et al. (2007) reported that the virus is detected during the autumn season in Taiwan. It is evident that the virus has no predilection to a particular season and is not affected by temperature variations as infections can occur throughout the year (Table 1).

There were 62 patients examined and tested in 2010 and 72 patients in 2015, respectively. Patients were one month to 16 years of age with median age 3 ± 3.34 years in 2010 and 3 ± 3.39 years in 2015. Overall, there were 56 girls (41.8%) and 78 boys (58.2%) with female-to-male ratio of 1 : 1.4 (1 : 1.3 in 2010 and 1 : 1.5 in 2015, resp.). Viral etiology was established in 109 out of 134 (81.3%) patients with ARI (54/62, 87% in 2010; 55/72, 76.4% in 2015, resp.). There were 43 (39.4%) female and 66 (60.6%) male infected children with female-to-male ratio of 1 : 1.5. Infected children's characteristics and characteristics of infection including number of pathogens detected, localization, and severity of disease for each season are presented in Table 1. There were no statistical significant differences between the two investigated seasons regarding the abovementioned categories (Table 1). Average length of hospital stay was 6.2 ± 5.4 days in both seasons.

A single virus was diagnosed in 61.3% (65/109) of the patients, coinfection with two viruses in 27.6% (30/109) of the patients, and concurrent detection of three viruses in 11.0% (12/109) of the patients. There were two cases of concurrent detection of four viruses (1.8%) one in each of the investigated seasons. There were no statistical significant differences between single and multiple virus infection regarding patients age (P = 0.0998), localization of infection (P = 0.3818), and severity of disease (P = 0.5147). However, some of the viruses were significantly more often detected in multiple infection combination than other viruses. These are AdV (P = 0.0013), HRV (P < 0.0001), PIVs (P < 0.0001), and HBoV (P = 0.0002). Table 2 presents the incidence of certain viruses and their representation in single infection and coinfection.

The most commonly diagnosed virus in both seasons combined was RSV (28.6%; 48/168), followed by PIVs types 1–3 (PIV-3 12.5%, 21/168; PIV-1 3.6%, 6/168; and PIV-2 2.3%, 4/168), HRV (14.3%, 24/168), HMPV (10.1%, 17/168), AdV (7.1%, 12/168), Flu A+B (4.8%, 8/168), and HCoV (4.2%, 7/168). However, incidence of viruses differs between seasons (Figure 2). There were no Flu viruses detected in 2010 and eight of them were detected in 2015 (P = 0.0014). PIVs were significantly more often detected in 2010 than in 2015 (P = 0.0001) with the highest frequency of PIV-3 detection in both seasons. Distribution among the types of PIV did not differ between the seasons (P = 0.4854).

In 2015, four additional viruses were tested, HEV, HBoV, PIV-4, and HPeV, revealing the following detection rate in 2015: HEV 13.2%, HBoV 10.5%, PIV-4 2.6%, and HPeV 1.3%, respectively.

The highest number of all viral infections was diagnosed in the 1–3-year-old group in both seasons (54.4%, 50/92 in 2010 and 38.2%, 29/76 in 2015) with observed differences in season's incidence of PIV and HRV in relation to the age of the patients (Figure 2). In children below one year of age, higher incidence of HRV and PIV in 2010 compared to 2015 was recorded (P = 0.0092 and P = 0.0092, resp.), and in children 1–3 years of age higher incidence of PIV in 2010 than in 2015 (P = 0.003) was also observed. RSV incidence was the highest in 1–3-year-old children in 2010 in contrast to the highest RSV incidence in <1-year-old group in 2015. However, there were no statistical differences observed comparing RSV incidence between the seasons in relation to patients age (P = 0.426 for <1-year-old group and P = 0.062 for 1–3-year-old group, resp.). HRV was significantly more often detected in children with URTI (P = 0.0082), while RSV was significantly more often detected in children with LRTI (P < 0.0001) compared to the other viruses.

In total, 590 patients were screened for respiratory viruses. The male:female ratio was 346:244 (1.42:1). The median age was 1.75 years (range 7 days to 17 years). According to the age groups, there were 210 (35.6%) patients in the group <1 years of age, 168 (28.5%) patients from 1-2.99 years of age, 68 (11.5%) patients from 3−4.99 years, and 144 (24.4%) patients with ≥5 years of age. Clinical diagnosis of URTI has been determined for 277 (46.9%) patients, and LRTI for 313 (53.1%) patients. Any type of respiratory virus has been detected in 451 (76.4%) patients; in 315 (69.8%) of positive patients there was a monoinfection with a single virus, while in 136 (30.2%) positive patients two or more viruses were detected concurrently.

The exact prevalence pattern of detected viruses can be seen in Table 1. RV was the most frequently detected virus, diagnosed in 197 patients (33.4%); 60.4% as monoinfection, and 39.6% as coinfection with other respiratory viruses. This was followed by RSV (19.3%), AdV (15.6%), PIVs (9.5%), Flu types A and B (7.6%), HCoV 229/NL63 and OC43 (7.1%), HBoV (5.3), HEV (4.6%), and HMPV (3.1%).

Although human coronaviruses are characteristically causing self-limiting short diseases, the question of potential chronic SARS infections is of major importance for a future disease control. If the SARS-CoV is able to cause a chronic persistent infection, chronic carriers may serve as sources for new SARS outbreaks. However, the detection of SARS-CoV in stool of patients for longer periods than 6 weeks after hospital discharge has not been reported so far. Therefore, the danger of chronic carriers may not be relevant. In contrast to common human coronavirus infections with short durations, most animal coronaviruses cause persistent infections. As an example, the feline coronavirus FIPV infects animals which then continue to shed virus for periods reaching up to seven months after infection without carrying disease symptoms. Also, TGEV and MHV tend to cause chronic infections as these viruses may be found in the airways and small intestine (TGEV) or the nervous system (MHV) several months after infection. Although the SARS-CoV has jumped to humans it may still have this property of inducing chronic infections. Thus, SARS-CoV RNA was found in patients' stool specimen more than 30 days after the infections.

There was a significant difference in age according to the specific virus (P < 0.001) (Figure 1). Median age in years of RV infected children (2.25, IQR 6.50) was higher than in children infected with RSV (0.41, IQR 1.29), PIVs (1.04, IQR 3.00), HCoV (1.33, IQR 9.16), HMPV (0.92, IQR 4.09), and HBoV (1.21, IQR 1.71), but lower than in those infected with Flu (3.58, IQR 5.59), AdV (2.88, IQR 4.44), and HEV (3.66, IQR 5.29) (Figure 1). There was no statistically significant difference in the prevalence of RV infection between age groups (P = 0.781). Additionally, age (1 year increase) was not significant predictor for RV positivity (OR = 1.01, 95% CI = 0.97–1.06; P = 0.534).

According to the clinical presentation, there was a significant difference in the proportion of LRTIs between the type of the respiratory viruses (P = 0.002) (Figure 2). More than half of children infected with RV (110; 55.8%) presented with LRTI; nonetheless, this was not significantly different from the proportion of RV positive children with URTI (87; 44.2%) (P = 0.336), while children with RSV infection significantly more often presented with LRTI (P < 0.001).

Epidemiological and clinical information on the study samples and subjects retained through the anonymisation process was used to compare seasonal and age distribution of HRV-C infections with those of other common respiratory virus infections (Figs. 2, 3). In contrast to RSV and influenza A virus which showed pronounced increases in incidence in winter months, HRV-C infections occurred year round with similar incidence in each calendar month investigated in the current study (Fig. 2A), more comparable to adenovirus. In contrast, HRV-C resembled RSV closely in targeting young children and infants, with peak incidence of both viruses occurring in the 4–6 month age range, with a younger age distribution than adenovirus infections and distinct from the wide age range of influenza A virus (Fig. 3). 85% of HRV-C infections were observed in children younger than 2 years of age. No systematic difference was evident between HRV-Ca and HRV-Cc in their seasonal distribution or age range of infection.

Detection frequencies of HRV-C and other respiratory viruses among subjects presenting with LRTIs, URTIs and those without respiratory symptoms were compared (Fig. 4). Subjects compared were those presenting acutely and excluded those with underlying immunosuppression or severe underlying disease (see Methods for criteria to assign disease categories). One or more respiratory virus was detected in 55% of subjects with LRTIs, 65% in URTIs and 22% in those with no relevant respiratory or other symptoms/diagnoses. Dividing these groups, HRV-C was the only virus detected in 6.9% of LRTI cases and in 7.8% in URTIs, higher than in the non-respiratory disease controls (3.7%) and second only to RSV in detection frequency (15.7% and 10.0% respectively). HRV-C was twice as frequently detected in subjects with respiratory disease than in those without relevant symptoms (p = 0.009 by Chi-squared test). HRV-C was detected in children in paediatric intensive care units (12%, 14 patients), mostly with pneumonia and without any other cause identified, in neonatal intensive care (11%, 4 patients). There was no significant difference between disease categories in the proportion of HRV-Ca and HRV-Cc variants.

Reintroduction of the Ad vaccine in basic training populations has again been extraordinarily successful in reducing the burden of both Ad-related respiratory illness and the burden of respiratory illness with fever. 75% reductions in FRI have been demonstrated by others, including at Joint Base San Antonio-Lackland where this study was conducted, against the backdrop of a 99.6% reduction in the weekly rate of Ad-related illness [11, 16]. Sustaining the commitment to prevention of Ad-related illness in uniquely susceptible trainees will be necessary if history is not to repeat itself. However, prevention of respiratory illness in this population is a complex task. Risk factors for transmission of respiratory pathogens will continue to be present in conditions inherent to basic military training. Adenovirus, despite its preeminence as a pathogen of interest in this group, has never been the entire story, and large outbreaks of non-vaccine serotype Ads have occurred even while vaccine serotypes were circulating. Influenza causes annual epidemics which, in the context of an effective vaccine program, are typically limited in this population, but which can have considerable impact when new strains emerge. In summer and fall of 2009, influenza was responsible for 20% of FRI in those who were tested [20, 21]. Large outbreaks of pharyngitis caused by S. pyogenes, complicated by acute rheumatic fever, pneumonia, necrotizing skin and soft tissue infections, and other suppurative and immunologic complications, have been reported throughout the past century, prompting widespread use of antimicrobial prophylaxis at training sites [22–24]. Pneumococcal outbreaks have also occurred despite such prophylaxis, including pneumonia and fatal meningitis [25, 26]. Others, including Neisseria meningitidis, Bordetella pertussis, M. pneumoniae, and C. pneumoniae, have been well-described in this population. Horizontal efforts at respiratory infection prevention, such as promoting hand hygiene, environmental including gas mask disinfection, cohorting of ill trainees, and respiratory etiquette, will require continued emphasis, even with near-elimination of Ad-related illness. However, vertical measures targeting specific organisms have also been demonstrated to have significant impact, with Ad vaccine as the prime example, and ongoing exploration into post-VI causes of illness will be necessary to direct further interventions.

Furthermore, although widespread efforts exist to monitor FRI rates and conduct surveillance for common respiratory viruses, not all acute respiratory illness is febrile. Clearly, trainees are still presenting for illness, but those without fever, which now represent >90% of those presenting for care, will not have respiratory pathogen analysis via DoD-directed surveillance mechanisms. Few prior data inform clinical differences between those presenting with Ad vs other respiratory pathogens. Recent comparisons of pdm(09)H1N1 influenza and Ad, including an analysis from this cohort, corroborated a predominance of coryza and cough presentations for influenza, vs pharyngitis for Ad [20, 21]. This evaluation again emphasizes a classic presentation of Ad-related illness: fever, systemic complaints, and pharyngitis, distinct from the afebrile, coryza/cough presentations of those presenting post-VI and with rhinovirus. Interestingly, documentation of abnormal lung examination findings increased post-VI, as did use of clinical diagnostic terms suggesting LRTI, including both bronchitis and pneumonia. It is likely that most of those labelled “pneumonia” were never confirmed with radiographs, and absence of fever with these argues against that diagnosis in this young, otherwise healthy population. The predominant organism identified among these was rhinovirus, which, while not classically associated with lower respiratory disease in healthy adults, has been described including within military training populations [28, 29]. Nevertheless, the combination of increased LRTI diagnoses, and the increase in cough as well as physical exam findings of the same, provide signal of an increase in LRTI post-VI which should be explored with targeted research. It is also considerable that, despite a relatively broad panel of respiratory pathogens targeted with molecular methods, >50% post-VI had no pathogen detected. Some of these may have been non-infectious, as suggested by the increase in clinical diagnostic terms relating to allergies, but this represents a significant research gap. The nature of respiratory illness itself in basic military trainees has changed after reintroduction of Ad vaccine, transitioning from a febrile pharyngitis marked by systemic signs and symptoms, to an afebrile, cough and coryza predominant illness.

The ecologic niche occupied by vaccine-serotype Ad in this population was remarkable, causing approximately 70% of all FRI historically and with 80% of trainees infected by the end of training [4, 11]. During Ad VI in 1971, molecular methods for pathogen surveillance were not available; despite this, serotype shift was observed. Initially, Ad4 was the only serotype included in the vaccine program, but Ad7 was later added after this emerged and replaced Ad4 as the predominant cause of FRI. Since that time, dozens of additional Ad serotypes and other respiratory viral pathogens such as bocavirus and human metapneumovirus have been identified. Respiratory pathogens cause outbreaks, which may come and go independently of a vaccine program's effect, so changes in frequency must be interpreted with caution. However, it is reassuring that Ad14 has not yet reemerged in this population and, in fact, decreased in frequency since VI, a finding which has been corroborated by others, and potentially related to cross-protection with Ad7 immunity [9, 16]. The decrease in frequency of influenza A was driven by the unusually high number of influenza A cases in 2009, which contributed 63 to the total of 76 during the entire study. The trend toward a decrease in S. pyogenes (with no changes in antibiotic prophylaxis during the study period) is potentially biologically plausible with viral coinfection increasing the likelihood of streptococcal illness, although specific associations between Ad and S. pyogenes have not been established, and rates of S. pyogenes illness are not known to have changed during the first iteration of the Ad vaccine program. The small increases seen in detection of M. pneumoniae, bocavirus and coronavirus OC43 may be due to chance alone or natural variation, but bear further observation. Most significant was the increase seen in detections of rhinovirus, which increased as a proportion of detected pathogens, in rates of positive tests among those tested for rhinovirus, and in raw numbers despite fewer overall enrollments. Rhinovirus may be associated with decreased probability of detecting other respiratory viral pathogens, including Ad. An evaluation of virus pairs in a PCR-based analysis of co-detections demonstrated a negative association between Ad and rhinovirus. While multiple additional respiratory viruses also demonstrated a negative association with rhinovirus, no others correlated either positively or negatively with Ad. A negative interaction between these 2 pathogens has previously been reported in military recruits with and without symptoms of respiratory illness, and with Ad, but not rhinovirus, clearly associated with illness.

The most significant strength of the study is the collection of a wide array of respiratory pathogen data alongside a detailed collection of clinical and demographic data, allowing thorough evaluations for ecologic niche replacement, as well as changes in clinical illness, throughout a major change in vaccine administration, in >2600 trainees. Others include the high uptake of Ad vaccine, and the consistency in access to care, living and training conditions, and preventive medicine measures throughout the study period. The study also has a number of limitations. This is a single center which, although spanning several years, cannot account for natural variability of respiratory pathogens here or at other training sites, and some pathogens were tested for only from a subset of samples. These represent a convenience sample of the overall burden of trainees presenting to medical care for respiratory illness. Trainees, for a number of reasons, may be reluctant to self-identify when ill and present for care. While we have no reason to believe this limitation changed over the course of the study, this limits the ability to extrapolate frequencies of detection to rates of disease. Since asymptomatic subjects are not captured, it also limits the ability to determine colonization vs correlation with clinical illness. However, study procedures and approaches to enrolling trainees were unchanged over the course of the study period. There may have been increases in healthcare-seeking behavior in 2009 during the influenza pandemic, and this year saw the highest number of enrollments over the course of the study. Finally, comorbidities in this group were not captured, although significant known comorbidities are typically disqualifying for military accession.

Soon after the identification of a new coronavirus as the causative agent of SARS and of a southern Chinese province as the first area of occurrence, animal species of this area have been speculated to be the origin of the SARS-CoV. As analysis of the SARS-CoV genetic sequence revealed large differences to any other currently known coronaviruses in humans or domestic animals, it was hypothesized that the new virus might originate from wild animals. This hypothesis was supported by a search for coronaviruses in wild animals sold on markets in southern China, which identified the presence of a coronavirus in civet cats. This animal coronavirus was shown to have a sequence identity of more than 99% to the SARS coronavirus with only a limited number of deletions and mutations between both viruses. SARS-CoV has a deletion of 29 nucleotides relative to the civet cat virus, indicating that if there was direct transmission, it went from the animal to man, because deletions occur probably more easily than insertions. Recent reports indicate that SARS-CoV is distinct from the civet cat virus and it has not been answered so far if the civet cat virus is the origin of the SARS-CoV or if civet cats were also infected from other species. Therefore, there are no data available on the possibility of horizontal transmission between animals, and the question whether the jump of the virus from an animal to humans was a single accident or may frequently occur in future with the animals as dangerous reservoirs for future SARS epidemics remains unanswered. So far, the SARS-CoV has been reported to be able to infect not only humans but also macaque monkeys, domestic cats, and ferrets. However, transmission of the virus from the domestic cat to man has not been shown. The ability of the SARS-CoV to infect other animal species could point to potential natural reservoirs of the virus. In this respect, coronaviruses are known to relatively easily jump to other species. I.e., the human coronavirus OC43 shares a high degree of genetic sequence homology to bovine coronavirus (BCoV) and it is commonly assumed that it has jumped from one species to the other. In the same way, BCoV has been reported to be able to infect humans and cause diarrhea. Whereas the precise mechanisms of these species jumps remain unclear, it is most likely that they represent the results of mutations and epidemiological studies of coronavirus infections in wild animals will therefore be crucial for future understanding and control of new SARS outbreaks.

The distribution of the viral etiologies of the four age groups is shown in Table 2. The highest proportion was observed in young children (75.0%), and lowest proportion was observed in adults 18–60 years of age (42.2%). Similarly, the positive rate of cases with a single virus infection was highest in the young children (65.5%) and lowest in adults of 18–60 years of age (38.5%). Moreover, the positive rate of the cases with coinfections was highest in older adults (11.9%), followed by young children (9.5%). The predominant viruses among four age groups differed. FluA virus and subtypes were the most prevalent viruses in the older adults (≥60 years), and the converse was true in the young children (< 5 years). EV, RSV and PIV viruses predominated in the young children (< 5 years), and FluB, RhV and ADV viruses were more prevalent in the young adults (5~18 years).

Of the 735 patients, 285 (38.8%) displayed evidence of viral infection, 163 (57.2%) of whom were males and 122 (42.8%) were females. Among the 285 patients, 331 viruses were detected because 44 (15.4%) patients were coinfected with two respiratory viruses and 2 (0.7%) were coinfected with three respiratory viruses (table 1). The most common viral infections were HRV 122 (42.8%), followed by Flu A 47 (16.5%), RSV 42 (14.7%), HCoV-OC43 25 (8.8%), AdV 22 (7.7%) and hMPV 15 (5.3%). The newly discovered viruses WUV, KIV, Boca and hMPV were detected in 43 (15.1%) of the patients with RTI (table 1).

Of the 285 patients, 167 (58.6%) respiratory viral infections were detected in infants aged <2 years (table 2). Among these infections, Boca was responsible for 11 (3.9%), hMPV for 7 (2.5%) and KIV for 3 (1.1%). Regarding the other viruses, HRV was responsible for 64 (22.5%), RSV for 36 (12.6%) and HCoV-OC43 for 11 (3.9%) infections. It was interesting to note that 20 (7%) of the Flu A infections were in patients aged >30 years. Overall, the majority of RTI, reaching 202 (70.9%), were among children ≤5 years of age (table 2).

The distribution of viruses detected in relation to hospital admission is shown in table 2. The majority of the patients admitted to wards had HRV infections (n = 68, 23.9%), followed by patients with Flu A infections (n = 35, 12.3%). Most of the children were admitted to the pediatric intensive care unit for HRV (n = 43, 15.1%), followed by RSV (n = 23, 8.1%). Furthermore, 11 (3.9%) of the patients with viral respiratory infection were admitted to the intensive care unit due to HRV, followed by Flu A (n = 6, 2.1%).

These viruses were most commonly detected in NPS (104 samples, 30.1%), followed by NPA (99 samples, 28.7%), BAL (87 samples, 25.5%) and TA (55 samples, 15.9%; table 2). Table 3 shows that the majority of the infections caused by the investigated respiratory viruses affected the LRT (251 infections, 88.1%) rather than the URT (80 infections, 28.1%). Pneumonia and bronchiolitis were the most frequent reasons for hospitalization (170 infections, 59.6%).

The prevalence of respiratory viral infections was highest during the period from October to February and then declined to the lowest level from July to September (fig. 1). The incidence of newly discovered viral infections was highest during the months of January, February and October. Positivity for WUV and hMPV peaked in February [3 (1.1%) and 7 infections (2.5%), respectively] and Boca peaked in March (5 infections, 1.8%). KIV presented consistent numbers from October to January (1 infection, 0.4%). The other respiratory viral infections were generally present throughout the year, reaching a peak during the winter months from October to February.

The Ethics Committee at IMIP and the National Research Ethics Office of Brazil approved the study and written informed consent was obtained from the parents prior to enrolment.

At the end of follow-up, 48 patients had been diagnosed with CLAD (Table 2). Of these, 11 were predominantly restrictive and 37 were predominantly obstructive. A total of 48 patients suffered organ loss, of which 42 patients died and 6 had a retransplantation. Twelve of the deaths occurred during the first year. The major cause for organ loss was CLAD (n = 25), and other significant causes were infections (n = 7) and malignancy (n = 6). Chronic lung allograft dysfunction development during the follow-up period was more common among the VRTI positive subjects (P = 0.005) (Table 2), and it was significantly more common that CLAD was cause of organ loss among those who had suffered from a VRTI (P = 0.008). We found no significant difference regarding graft survival at the end of follow-up between patients with and without VRTIs (P = 0.84) (Table 2). The VRTI-positive subjects had a significantly higher hazard ratio (HR) for CLAD development in multivariate analysis (P = 0.041) (Table 3), but we found no significant difference in time until graft loss (P = 0.86). However, among those who suffered from organ loss, time between CLAD development and organ loss were significantly longer for those who had 1 or more VRTI before developing CLAD, compared with those who had none (P = 0.021). Bacterial (P = 0.013) and fungal (P = 0.001) infections were associated with shorter time to graft loss.

In total, 26 patients suffered an AR during the first year, 3 of these patients suffered more than 1 AR before developing CLAD. Bacterial infections affected 41 patients and consisted of Pseudomonas aeruginosa in 20 cases, and the remainder were other bacteria. Fungal infections affected 13 patients with 2 cases of Candida glabrata, 1 case of Candida krusei and the rest consisted of Aspergillus species. A total of 14 patients showed CMV antibody mismatch; 46 patients had at least 1 episode of CMV viremia during the first year, but only 4 patients developed CMV disease during the follow-up period. Repeated elevated EBV-DNA was common, with 29 patients suffering from more than 1 episode of Epstein-Barr viremia. Three patients developed posttransplant lymphoproliferative disease during follow-up, all responded favorably to treatment. Acute rejection and infectious events are outlined in Table 2. No case of positive PCR for Legionella pneumophila, Pneumocystis jirovecii, Chlamydophila pneumoniae, or Mycoplasma pneumoniae was detected.

Of 36 influenza PICU patients with underlying chronic conditions, four (11.1 %) were too young (<6 months of age) to have been immunized against influenza, and for two patients (5.6 %), data on their influenza vaccination status was unavailable. Twenty-nine (80.6 %) patients from this risk group had not been vaccinated against influenza although they would have been eligible. One immunocompromised child (2.8 %) had been vaccinated in October 2012, but was diagnosed with A(H3N2) in January 2013.

“Surveillance and early warning systems” should be set up in emergency conditions. This involves watching diseases on a continuum, finding trends, and reporting outbreaks earlier rather than later. This is a data collection phase, similar to the rapid assessment phase, but on a larger scale, and primarily involves interpretation of the data collected in order to create an efficient and effective public health response to the threat.

Underlying chronic medical conditions were reported for a total of 36 influenza PICU patients (76.6 %) (Table 3). Chronic neurological diseases were most frequent (34.0 %), followed by chronic lung disease (25.5 %), preterm birth (21.3 %), cardiac malformations (17.0 %), obesity (10.6 %), genetic disorders (8.5 %), and immunocompromising conditions (8.5 %).

We report the rare finding of BCG reactivation in a child with confirmed measles infection. Although BCG reactivation is known to be an extremely important and highly specific clinical manifestation of KD, this case suggests that BCG reactivation may also occur in other childhood diseases or infections. A thorough search for an infective aetiological agent may be indicated in children presenting with this clinical finding.

HPIVs are common respiratory pathogens and are important causes of URTI and LRTI. Previous studies have predominantly focused on HPIV-1, HPIV-2 and HPIV-3 infection in children because of high positive rate and morbidity of three types of HPIV infection in children, therefore less is known about HPIV-4 infection and HPIV infection in adults. In this study, we analysed the characteristics of the four HPIV types in children and adults with ARTI in Guangzhou, southern China over a 26 month period.

Of the pathogens investigated in this study, HPIVs were the sixth most frequently isolated. The predominant types were HPIV-3 and HPIV-1, which is consistent with previous reports. HPIVs were isolated with higher frequency from males than females, similar to the previous study. Immunity to HPIVs is incomplete, and infections occur throughout life. In this study, HPIVs were detected in patients over a wide age distribution. However, many more children were infected than adults (p<0.001), and the vast majority of HPIV infections occurred in patients under 5 years of age. The four HPIV types differed in age distribution of patients infected. HPIV-3 was mainly detected in paediatric patients under 3 years old, while HPIV-1 and HPIV-2 were isolated from a broader age distribution than HPIV-3 (Figure 1). No infant under one month of age was HPIV positive. These results are in accordance with seroprevalence studies indicating that newborn infants have high levels of HPIV antibodies, and that these levels decrease substantially by 7 to 12 months of age. In this study, HPIV-4 was only detected in children (4 months to 8 years old). This result is different from previous studies in which fairly equal infection rates were reported among infants younger than one year old, preschool children, school age children and adults. The low HPIV-4 positivity in our study may have influenced our results.

Co-infection of HPIV with other respiratory pathogens was common for all four HPIV types, similar to previous reports. RSV, MP, EV and HCoV-OC43 were the main co-detected pathogens in this study. Co-infections were mostly found in children, especially in patients under 5 years of age (Figure 1). This might indicate that immature immune systems of children leave them susceptive to potential pathogens. However, no significant differences in clinical presentation were seen between patients solely infected with HPIV and patients co-infected with HPIV and other respiratory pathogens (Table 2).

Biennial fall epidemics of HPIV-1 have been reported in previous studies. HPIV-2 has been reported to cause infections biennially with HPIV-1, to alternate years with HPIV-1, or to cause yearly outbreaks. HPIV-3 is reported to have occurred annually during April to June in the United States. Only a small number of studies have studied the epidemiology of HPIV-4, and the numbers of infections were too low to clearly identify seasonal peaks in activity. In this study, HPIVs were isolated throughout the year. Seasonal peaks of HPIVs, driven mostly by HPIV-3 and HPIV-1, occurred in the time when autumn turned to winter and summer turned to autumn (September 2009 to November 2009; September 2010 to October 2010; April 2011 to July 2011). These results differ from previous reports. In addition, HPIV-3 and HPIV-1 did not show the competitive interaction described previously, where HPIV-3 activity appeared to be greater during years when HPIV-1 was not circulating. The different geographic location might lead to the different seasonal distributions of HPIVs. HPIV-2 and HPIV-4 were not isolated as frequently as HPIV-3 and HPIV-1, and the seasonal peak of HPIV-2 and HPIV-4 was in the turn of winter to spring (December 2010 to March 2011). The frequency of detection of these two HPIV types increased when detection of HPIV-3 and HPIV-1 declined.

HPIVs can cause a spectrum of respiratory illness, and more “hoarseness” (p=0.015), “abnormal pulmonary breathing sound” (p<0.001), “dyspnoea” (p<0.001), and “pneumonia” (p=0.01) presented in HPIV-positive patients than HPIV-negative patients. In our study, more “diarrhoea” (p<0.001) presented in HPIV-positive patients than HPIV-negative patients suggesting pathogenic activity of HPIV in gastrointestinal illness (Table 2). Systemic influenza-like symptoms were not main presentations of HPIV-positive patients overall (Table 2), but HPIV-positive adult patients presented with significantly more systemic influenza-like symptoms than HPIV-positive paediatric patients (p=0.005). This result suggests that the clinical presentation of HPIV infection may differ by patient age as previously shown for HBoV.

The relationship between HPIV infection and neurologic disease has been studied for many decades. In a previous report, children hospitalized with HPIVs had serious febrile seizures. In contrast, we found no significant difference in convulsion between HPIV-positive and HPIV-negative patients (p=0.726).

This study investigated presentations of the four HPIV types and attempted to distinguish between different types of HPIVs by clinical presentation. However, because all four HPIV types caused a wide spectrum of symptoms, distinguishing HPIV types by clinical characteristics alone was not possible as the previous study.

Evidence for significant interactions between respiratory viruses was obtained through comparisons of detection frequencies in mono- and doubly-infected individuals (Fig. 5). HRV-C was nearly three times more commonly detected in those co-infected with adenovirus (16.0%) than in mono-infected subjects (5.8%; p<0.001). Examples of negative interactions were additionally observed. Most strikingly, HRV-C had a highly significant effect on detection frequencies of RSV and other respiratory viruses. RSV mono-infection was found in 10.7% samples but virtually absent among HRV-C infected subjects (2.4%, p<0.001). For other respiratory viruses (excluding AdV), a 3.5-fold reduction in their infection frequency was observed in HRV-C co-infected subjects (12.9% to 3.7%; p<0.001). This reduction was greater than the effect of RSV co-infection on their detection frequency (<2-fold). In contrast, RSV and the group of other respiratory viruses did not significantly influence the likelihood of HRV-C detection; study subjects showed a 5.8% HRV-C mono-infection frequency compared to 4.6% and 8.6% in those co-infected with RSV and other respiratory viruses respectively (p>0.05). Together these observations indicate that it is HRV-C that is inhibiting RSV and other respiratory infections rather than the converse.

Many groups have reported that the occurrence of co-infections with HCoV-NL63 and other respiratory viruses, including other human coronaviruses, influenza A virus, respiratory syncytial virus (RSV), parainfluenza virus and human metapneumovirus (hMPV), are common [26, 30, 46, 47, 51, 53, 54, 60, 61]. Also, co-infected patients are more likely to be hospitalized, indicating the severity of this kind of superinfection. In a study from Germany, RSV-A and HCoV-NL63 was the most common co-infection indentified in children less than three years of age. This is probably due to the high incidence of RSV-A in winter and the overlap in seasonality of the viruses. Also, in Italy, HCoV-NL63 circulates as a mixture of variant strains and is often associated with other viral infections. In South Africa, co-infection of patients with HCoV-NL63 and bocavirus in hospitalized children is reported. Nasopharyngeal and bronchoalveolar lavage samples from 341 patients were screened for common respiratory viruses, and the co-presence of HCoV-NL63 and bocavirus in at least one sample was reported.

Interestingly, the viral load of HCoV-NL63 is lower in co-infected patients than in patients infected with HCOV-NL63 only. There are various possible explanations for this phenomenon:

The high prevalence of co-infections of HCoV-NL63 and other respiratory viruses increases the chances of genetic recombination with these human or zoonotically transmitted viruses. In fact, Pryc et al. (2006) states HCoV-NL63 resulted from a recombination event between PEDV and an ancestral HCoV-NL63 strain. Theoretically, these types of recombination events could enable highly pathogenic virus variants to arise.

One hundred-three patients under nine years of age hospitalized at the Pediatrics Department were enrolled. There were 61 males and 42 females with a male/female ratio of 1.4 to 1. The characteristics of patients are shown in Table 1. The mean age of patients was 34.9 (1–106) months, where 89.4% of patients where 5 years of age. Among the patients 18.4% were <12 months; 32% were 13–24 months of age, 39% were 25–60 months, and 10.6% were 61–106 months (Table 1). There were no statistically significant differences among age groups.

Among the patients, 42 had other significant comorbidities (e.g. chronic bronchitis, other chronic diseases and prematurity). Thirty-four patients (33%) were attending school and daycare centers.