Overall, 291 of the 4337 vaccinated individuals returned the questionnaire and reported adverse reactions. This is a rate of 6.7%. The majority of reported adverse reactions was found in the age between 30 and 39 years. (Figure 2)

The most frequently reported local site reactions were: local pain/pruritus or the sensacion of heat at the injection site in 3.8% out of the 4337 vaccinations, myalgia or arthralgia in 3.7%, induration or erythema at the injection site in 2.6%, lymph node swelling in 0.9%, skin rash in 0.3% and ecchymosis at the injection site in 0.1% (Figure 3).

The presence of systemic adverse reactions were reported as follows: fatigue in 3.7%, headache in 3.1%, flu-like symptoms in 2.3%, shivering in 1.9%, temperature > 38°C in 1.3%, dizziness in 1.1%, increased perspiration in 1.1%, gastrointestinal symptoms in 1.0%, drowsiness in 0.9%, , insomnia in 0.7%, formication in 0.3%, Further some severe reportable adverse reactions were observed (0.5%, Figure 3) as one case of facial nerve paralysis, one case of rheumatoid arthritic symptoms and one case of skin alteration which was reported to the local health authorities and the Paul-Ehrlich-Institute.

We classified the admitted patients into 4 age groups, as mentioned previously. Interestingly, there were significant differences in the male-to-female ratios in the admitted patients and the pneumonia patients between children (≤ 15 y) and adults (16-86 y). In the admitted patients, the male-to-female ratio was 1.6:1(132:84) in children, whereas the ratio was 1:1.4 (64:92) in adults. Additionally, in the patients with pneumonia the male-to-female ratio was 3:1(60:20) for the children and 1:2(12:24) in the adults. There was a correlation between increased age and an increase in the admission rate and the frequency of underlying diseases (linear by linear association test), and the total duration of fever and the hospitalization (ANOVA test). In laboratory findings, the C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) values displayed similar correlation (ANOVA test) (Table 2). The ≥ 65 y group showed significant differences compared to other age groups in nearly all of the examined parameters (χ2 test and Student's t-test, data not shown). The frequency of underlying diseases was significantly different among age groups. Many patients had more than one underlying diseases and these patients were more prevalent in older groups. The underlying diseases of the admitted patients are shown in Table 3.

We previously found that children with pneumonia had higher leukocytes counts with lower lymphocyte differentials than the children without pneumonia. The adult patients with pneumonia had higher leukocytes counts, CRP and ESR values with lower lymphocyte differentials than the adults without pneumonia (Table 4).

We examined whether ILI symptoms segregated with the flu vaccination records. 58.0% of study participants who were vaccinated also reported the experience of ILI symptoms. 65 study participants who received the pdm flu vaccine and filed their ILI symptoms report later during the flu season (i.e. after December 2009, and after they had received the pdm flu vaccine) experienced significantly more symptoms than the 170 study participants who did not receive the pdm flu vaccine (and also reported their ILI symptoms after December 2009). Vaccinated study participants reported 4 ILI symptoms rather than 3 symptoms on average, i.e. nasal discharge (p = 0.02), fatigue (p = 0.04), cough (p = 0.01), sneezing (p = 0.04) and chills (p = 0.05) (Table 3). The 45 study participants who received the pdm flu vaccine and mailed a swab later showed less frequent detection (6/45) of coronavirus RNA (p = 0.03), but showed a higher percentage (14/45) of rhinovirus RNA (p = 0.01) in their nasal swabs as compared to the 139 study participants who chose not to get vaccinated but mailed a swab due to symptoms of ILI (44/139).

The median age of the 373 hospitalized patients was 10 y (range 2 mo- 86 y, mean 19.4 ± 20.1 y), and the male-to-female ratio was 1.1:1 (196/177). The total admission rate due to influenza infection was 8.4% (373/4,463). The age distribution of the patients admitted to hospital are shown on Figure 1 (black bar), and these demonstrated similar patterns to those of the total patient cohort except older age group. The admission rate of the 0-10 y was 8.8% (191/2,160), the 11-20 y was 4.7% (62/1,329), the 21-30 y was 9.2% (41/447), the 31-40 y was 8.9% (23/258), the 41-50 y was 12.1% (15/124), the 51-60 y was 16.7% (14/84), and the ≥ 61 y was 44.3% (27/61).

All inpatients received the recommended doses of oseltamivir and the majority of inpatients received a broad-spectrum antibiotic. In total, 338 patients (90.6%) received oseltamivir within 48 h of fever onset. Six adult patients (4 in the 41-65 y group and 2 in the ≥ 65 y group) with underlying diseases were infected during their hospital stay and these patients were excluded from the subjects. Additionally, four of the infected patients were pregnant women, and their clinical course was uneventful. We analysed the chest radiographs of the inpatients and found that 116 patients had pneumonia (80 children and 36 adults). Pneumonia was detected in 31.3% (116/373) of the admitted patients, and 2.6% (116/4,463) of the total infected patients, respectively (Table 1). No children were treated in the intensive care unit. However, six adult patients were treated in the intensive care unit, two had ARDS and 4 were at risk of deterioration because of underlying diseases. None of the infections in this study was fatal.

The mean duration of symptoms lasted 3.5 days, the maximal duration of symptoms was reported with 40 days.

RT-PCR is considered to be one of the most sensitive and specific tests for the diagnosis of influenza. In addition, multiplex PCR methods can detect a panel of respiratory viruses or co-infections simultaneously. In this study, pharyngeal swab specimens were collected from ILI case-patients, who strictly complied with Chinese CDC standards.

The general population in Beijing, China has been exposed to the novel pandemic H1N1 influenza virus since mid May 2009. According to the Chinese CDC, as of May 2011, 136869 confirmed cases and 875 deaths from pandemic influenza H1N1 2009, have been reported nationwide in mainland China,.

In this study, approximately 29.4% of the samples were positive for at least one virus, which is consistent with the results of other studies, in which between 0.9–27%, and 44% of reported samples were positive. Our research suggests that FLU-A was the predominant viral pathogen among ILI patients in Beijing from August 2010 to May 2011, which was similar to that of 2009, 2008 and 2006, but different to 2007,,,. Almost all positive samples demonstrated FLU-A strains, while few FLU-B strains were detected. This result differs from that reported from the United States, which demonstrated that 26% of the positive specimens were influenza B viruses. This discrepancy may in part reflect the epidemic of influenza A virus in North China in 2010–2011. It is possible that FLU-B may not have caused ILI symptoms severe enough for the sufferer to seek medical attention. It is also possible that FLU-B was not circulating in this geographical area during this time. According to the CNIC, Influenza B virus-positive rate was about 1.6% in North China from June 2010 to May 2011. The percentage of tests that were positive for influenza which included FLU-A and FLU-B was 23.7% which was lower than the same period in 2009,.

FLU strains, based on our data, accounted for approximately 80% of the RT-PCR positive cases, and in August, September and October 2010, and Mar 2011, all infections were caused by FLU-A alone. The incidence of FLU-positive specimens was high. This may reflect the possibility that our sample collection was biased towards patients exhibiting ILI, which is a clinical or symptomatic definition of influenza to identify potential influenza cases, in other word, influenza-like illness case-definition make influenza viruses as the virus most commonly detected,,,. A study of human-to-swine transmission of pandemic influenza A virus concluded that the human ILI case definition has a high specificity and a low sensitivity for FLU-A. Influenza viruses usually account for a much greater proportion of positive specimens of influenza-like illness in adults than other respiratory viruses during the peak seasons.

A total of 7 samples (8.5% of the total number of RT-PCR positive cases) revealed the presence of co-infections. In five FLU-A-positive samples, viral co-infections were observed, including one co-infection with HRCV in July, one with HRV in November, one with HRSV-A, and one with HRSV-A and HRV in January, and one with HRSV-A and HRV in February. A population challenged by multiple infectious agents may result in an epidemic and restructure of various viruses.

Rates of ILI are an indicator of trends for influenza pandemics. Beijing is located in the temperate zone of the Northern Hemisphere, where influenza typically peaks seasonally once a year, and Beijing experience one peak of influenza activity and the peak occurred during December–January next year before 2009,,, but ahead to November in 2009,. That the peaks in ILI and the increase in acute respiratory infections (ARI) are due to influenza is supported by the seasonal pattern of high-probability ILI, the low level of respiratory syncytial virus infections, and laboratory results in the influenza season,,. During the 2010–11 influenza season, a seasonal pattern in ILI activity was observed and influenza activity peaked in late January 2011 in Beijing. Compared with the previous pandemic year (2009), lower outpatient numbers were observed during 2010–11(Figure 2). Overall, the rates of influenza-like illnesses in outpatients were lower during the 2010–2011 season, than during the 2009–10 pandemic influenza season (Figure 3).

As a result of the requirement for fever in our definition of ILI, our calculated incidence may underestimate the true incidence of ILI in the cohort. Our study shows that the positive rate of influenza virus was consistent with changes in the ILI rate during the same period.

More than 70% of ILI-case patients were infected with p-H1N1 between June 2009 and January 2010. Compared to the previous year, the age of FLU-A patients ranged from 18 to 61 years with a mean age of 36.7 years, which is older than the age of 2009 ILI-case patients (mean age was 23.4 years).

In 197 (70.6%) specimens, no viral etiology was identified or the virus was not detected. This may reflect the fact that only viruses known to cause respiratory symptoms were tested, and therefore the remainder may have been caused by other respiratory viruses or by other micro-organisms, which could also have been additional pathogens in the positive specimens. It is also possible that some viruses could not be detected due to low levels of shedding.

In this study, we attempted to identify symptoms associated with influenza A infections. However, it is widely known that a clinical diagnosis of influenza is not straightforward, and it is difficult to find symptoms or combination of symptoms specific for influenza,. We found that cough was significantly associated with influenza A (χ2, p<0.001). This is consistent with the results of Boivin, of Ohmit and of Monto, who reported that cough and a fever>38°C were associated with a positive PCR test in the influenza population, when influenza was prevalent within the community,,. Our specimens were collected during the influenza season in Beijing and the cough appears to be specific for influenza in this cohort. This is also consistent with the view of Call et al, who believe that both fever and cough are more specific for influenza among elderly individuals when influenza virus is circulating in an area. We did not find any statistically significant difference in the occurrence of fever (temperature >39°C) between the FLU-A-positive group and the FLU-A-negative group (χ2, p>0.05). A study that involved all age groups demonstrated that muscle and joint pain and headache were associated with influenza. However, we did not find any statistically significant differences in the occurrence of muscle and joint pain, and headache between the FLU-A-positive group and the FLU-A-negative group (χ2, p>0.05).

This study has limitations. No testing for other etiologies of acute respiratory illness was performed. As is generally known, respiratory viruses, bacteria and other micro-organisms can cause respiratory illness with influenza-like symptoms. Without doubt, other micro-organisms could have been additional pathogens in the positive specimens.

In conclusion, pH1N1 did not affect typical influenza seasonal peaks, although FLU-A remained the predominant virus in Beijing in 2010. Symptomatically, cough was associated with FLU-A infection. The positive rate of influenza virus was consistent with changes in the ILI rate during the same period and there was a significant reduction in the incidence of ILI in 2010 compared to 2009. The findings of this study may facilitate the clinical discrimination of influenza A virus infection, as well as providing data and distribution information for virologic surveillance of influenza.

Anti-viral drugs are thought to be backbone of a management plan of an avian flu pandemic. Only two anti-viral drugs have shown promise in treating avian influenza: oseltamivir (Tamiflu®) and zanamivir (Relenza®). A treatment of Tamiflu® includes 10 pills taken over five days while Relenza® is administered by oral inhalation. The US Food and Drug Administration has approved both anti-viral drugs for treating influenza but only Tamiflu® has been approved to prevent influenza infection. Because antivirals can be stored without refrigeration and for longer periods than vaccines, developing a stockpile of antivirals has advantages as part of an overall strategy to control a flu epidemic. However, there are limitations to the use of antivirals: Tamiflu® needs to be taken within 2 days of initial flu symptoms for it to be effective, but many people may not be aware that they have the flu early in the disease. Some research in animals and recent experience in the use of the drug to treat human cases have also found that Tamiflu may be less effective against the recent strains for the current H5N1 virus than the 1997 strain. Improper compliance to antivirals by irresponsible individuals during an outbreak may results in the emergence of a drug-resistant strain. Lastly, there are current concerns about the safety of Tamiflu® which has been associated with increased psychiatric symptoms among Japanese adolescents.

We followed the study participants who gave blood at time points A and C and reported (in the spring of 2010) their 2009–2010 pandemic vaccination status. At time point A, the levels of A/H1N1/California/7/2009-specific Abs and IFN-γ production in response to the flu antigens were comparable between participants who received (after time point A) the pdm flu vaccine and individuals who chose not to receive the vaccine (seropositive at SRH > 4 mm2, but below protective levels (SRH < 25 mm2)).

At time point C, the levels of A/H1N1/California/7/2009-specific Abs were above protective levels (SRH ≥ 25 mm2) in pdm flu vaccinated participants, the Ab levels were significantly higher (p < 0.001) than in non-pmd flu vaccinated study participants. We also observed a statistically significant increase in IFN-γ production in response to all flu antigens (except for B/Malaysia/2506/2004), including the flu matrix antigen M1, in blood from study participants who received the pdm flu vaccine in the winter of 2009–2010 compared to the non-vaccinated study participants (p ≤ 0.04) (Table 2; note that M1 is not a designated component of the flu vaccine).

In respect of vaccination, the cases were more heavily influenced by others’ opinions and actions than were the controls. When compared, more cases would ‘accept advice from health professionals’ (OR2.67, 95 % CI 1.19-5.99, p = 0.02); and ‘family members who had received the flu vaccine’ than the controls (OR2.47, 95 % CI 1.54-3.95, p < 0.001).

External factors refer to unpredictable environmental factors, such as the occurrence of disease epidemics like Severe Acute Respiratory Syndrome (SARS) or pandemic influenza. High percentages of both cases (94.8 %) and controls (81.3 %) would receive a vaccine when there was a disease epidemic. When compared, more cases would receive a vaccine during an epidemic and the OR was 2.40 (95 % CI 1.07-5.37, p = 0.03).

A decrease in the number of patients seeking medical attention was observed in 2010 (Figure 2). In 2010, the overall level of outpatient consultations was less than that of 2009, particularly between May and December. From May 2009, when pH1N1 emerged in Beijing, the capacity of the outpatient service of the Infectious Diseases Department of PKUPH increased rapidly. The peak in numbers attending the outpatient department occurred in November 2009. From then, the outpatient volume declined, reaching normal levels in the third month of 2010. The numbers of outpatients remained relatively stable in 2010. The volume again increased during November and December 2010, and peaked in January 2011.

In addition, a significant reduction in the percentage of outpatient consultations for ILI was detected during 2010–2011 in North China (Figure 3). During the 2009 pandemic, the highest weekly ILI rate was 12.1 cases per 100 consultations in week 44 of 2009 (Figure 3). The ILI rate was relatively stable, based on data available from the weekly ILI surveillance system of North China in 2010. The percentage of patient visits for ILI peaked at 5.0% in late January 2011.

Currently, the H5N1 avian flu virus is limited to outbreaks among poultry and persons in direct contact to infected poultry. Avian influenza (AI) is endemic in Asia where birds often live in close proximity to humans. This increases the chance of genetic re-assortment between avian and human influenza viruses which may produce a mutant strain that is easily transmitted between humans, resulting in a pandemic. Unlike SARS, a person with influenza infection is contagious before the onset of case-defining symptoms. Researchers have shown that carefully orchestrated of public health measures could potentially limit the spread of an AI pandemic if implemented soon after the first cases appear. Both national and international strategies are needed: National strategies include source surveillance and control, adequate anti-viral agents and vaccines, and healthcare system readiness; international strategies include early integrated response, curbing disease outbreak at source, utilization of global resources, continuing research and open communication.

In this study, a comparison of the clinical features presented in children admitted to our hospital resulted positive for FLUA and B infections was analyzed. Bambino Gesù Children’s Hospital is a Reference Pediatric Centre for the care and treatment of children coming from central and southern Italy.

FLUB was slightly more prevalent (53.38 %) than FLUA (46.62 %) in the study population. This is consistent with the 2012–2013 ECDC Surveillance Report that reported a similar proportion of seasonal FLUA and B in Europe. However, FLUA peaked and declined slightly before FLUB and the highest infection frequency was evident during the “winter season”. Our data show a similar seasonal trend for FLUA and B; however, in contrast to the data reported by the ECDC, the circulation of FLUA ended later than FLUB.

Our results were consistent too with the prevalence of FLUA/H1N1 (34 %), FLUA/H3N2 (5 %), and FLUB (58 %) in Italy during the 2012–2013 flu season reported to Influnet, a sentinel surveillance system for influenza. Influnet also reported 3 % of FLUA cases that were not subtyped. However, these data reflect the whole population and our data are concerned with only pediatric patients.

The results of laboratory testing and clinical findings were compared for FLUA and FLUB patients to investigate clinical differences between the groups. No consistent differences were observed in the clinical presentation of patients by subtype viral, according to results of studies about the clinical characteristics of patients positive for Influenza A and B. Males seemed to be more susceptible to contracting FLU compared to females and, specifically, FLUB. This is concordant with previous studies in which the greater humoral and cell-mediated immune responses of females to viral antigens was demonstrated to play an important role in determining the gender variability in viral infections and, in females, being beneficial against infectious diseases. With regard to the age distribution, FLUA was more common in children less than 1 year old, where they can cause more severe infections, confirming previous studies [20, 21], and FLUB was more common in school age children.

Moreover, length of stay in children with FLUA was significantly higher than those infected with FLUB, being the median 5 days (range: 0–59) for FLUA and 3 days (range: 0–116) for FLUB. Specifically, this long hospital stay (59 and 116 days) was, for FLUA, in a patient with complications due to tracheostomy procedure, while for FLUB a nosocomial infection occurred in a oncoematological patient. This finding is unsual since that in a previuos study children with influenza B in comparison with those infected by A/H1N1 influenza virus had significantly higher hospitalization rates (p < 0.05). Is possible to speculate that the longer hospitalization for FLUA patients be correlated to the major cases with fever >38 °C and respiratory simptoms. No significant differences were observed in CRP levels between FLUA and B patients and a similar frequency of patients with elevated CRP was detected in both FLUA and B. Fever was confirmed as a major influenza symptom [21, 22]. A similar number of FLUA and FLUB patients presented with a temperature ≥38°. However, FLUB patients were significantly more likely to have a fever <38 °C than FLUA patients. In contrast, respiratory symptoms were mainly detected in FLUA patients (Table 1). Pre-existing diseases condition the clinical expression of Influenza resulting in a greater number of these children developing lower respiratory tract infection. We found that FLU patients with underlying malignancies such as lymphoma, leukemia, solid tumor, or tubulopathy, were more susceptible to FLUB infection, in agreement with data previously reported. In contrast, patients with congenital diseases (hypoplastic left heart syndrome, neuromotor disorder, Swyer James syndrome, epilepsy, cerebral palsy, laryngotracheal cleft, or immune deficiency) and genetic preexisting conditions (cystic fibrosis, sickle-cell anemia, trisomy of chromosome 10, or Niemann Pick disease) were more susceptible to FLUA infection. This is probably related to FLUA patients younger than those with FLUB. In our study, 12/133 (9.02 %) of FLU infections were hospital acquired with FLUB being the principal etiological agent responsible. Patients that contracted nosocomial infections were mainly immunosuppressed and admitted through the IIFU (8 cases FLUB), followed by the pediatrics (2 cases FLUB and 1 FLUA) and surgery (1 case FLUA) units. Numerous examples of nosocomial FLU outbreaks have been reported in long-term care facilities for the elderly. Experimental evidences supports the fact that humans generate infectious particles in both respiratory droplets and aerosols and that their generation is enhanced during influenza illness. Moreover, children, who do not have or minimal immunity against influenza viruses, and immunocompromised individuals, who can shed virus for long periods of time at high titers, have already been pinpointed as good transmitters in comparison to healthy adults. However, it is unclear why FLUB, in comparison to FLUA, should cause a more severe infection in the group of immunosuppressed and moreover, in the population object of the study, the number of patients with malignancies is low and it is difficult to establish if the difference is really significant.

In our study there were no significant differences between co-infections with viral or bacterial pathogens in FLUA and B patients. All of the co-infections investigated were in children with a mean age less than 5 years.

One death was reported in a FLUA patient affected by quadriplegia and chronic respiratory failure. It is possible that FLUA could have been responsible for further impeding the patient’s ability to breathe.

There were some limitations in this study. Being a retrospective study, it was not possible to collect more clinical data, as well as the information about the vaccination history of patients that tested positive for FLUA and B; in addition, this was a single-center study and only one year was analysed.

Our study was primarily focused on the clinical presentation of FLUA and FLUB infections to provide additional information concerning the clinical presentation of pediatric influenza. No outcomes due to other respiratory viruses were evaluated. Further studies that describe how co infections with other viruses impact FLU infections could be useful. To our knowledge, there is limited published data regarding the clinical differences between seasonal FLUA and B in pediatrics after the 2009 pandemics. The main findings from our study confirm that, although fever is a major component of influenza A and B presentation, respiratory symptoms were more severe and the length of the hospital stay was longer for FLUA patients than FLUB patients concluding that the increasing of FLUB in the season 2012–2013 was without any dramatic change in clinical manifestation. Although some studies have reported gastrointestinal symptoms such as abdominal pain, diarrhea and vomiting to be more common with FLUB infection, this was not the case in this study. The different viral types and subtypes should be routinely identifed by diagnostic laboratory to best address clinicians to appropiate therapeutic measures.

In summary, our results suggest that the clinical features correlated to different FLU viruses and to the relevant subtypes should be taken into consideration by health authorities to implement prevention strategies with the aim to reduce the number of sick subjects, the prevalence of hospitalization, and the circulation of FLU.

The analysis was limited to viral mono-infections and future studies should explore co-infections and bacterial infections. This study involved predominantly young adult males, and results may not be generalizable to the overall population, necessitating further studies among various age groups and gender. There were also less recruits amongst controls than amongst other groups, and this would be an important consideration when comparing the two groups in the future. Finally, the actual clinical impact of differentiating between various viral etiological agents may be limited, and we could not determine the relative severity of symptoms other than fever.

Our study highlights the varied etiology for FRI and ILI in the tropical setting – influenza and ADV and CV were all common. Influenza and ADVs tend to present with higher fever, and vaccination should be considered. The utility of ILI for tropical surveillance of influenza needs to be reviewed given the low PPV and high NPV compared to temperate regions. The surveillance system has enabled the Singapore military to understand the etiologic agents affecting servicemen, hence implementing and evaluating controls measures such as vaccination.

ARIs as the most common cause of morbidity and mortality in children, remaining a major concern, especially affecting children under 5 years old from low-income countries [1–4]. Unfortunately, information regarding their epidemiology is still limited in Peru [7, 11].

In recent years, there has been evidence of pertussis resurgence in Latin America, despite the introduction of the vaccine [14, 27, 28]. This bacterium is highly contagious and virulent, about half of the infected children under one year of whooping cough require hospitalization. In Peru, the national immunization program administers the combined pentavalent vaccine (DPT, HvB, Hib) at 2, 4, 6 months with reinforcements at 18 months and 4 years [29, 30]. Thus, we conducted a previous study on Peruvian children with a probable diagnosis of Pertussis and reported a Bordetella pertussis prevalence of 39.5%. However, the classical presentation of pertussis has proven to be not enough to achieve a definitive diagnosis and laboratory tests are of the utmost importance for etiological confirmation to avoid overdiagnosis [28, 31, 32]. In the light of possible coinfections and more than 60% of patients without an etiological identification in our previous study, we conducted a new comprehensive analysis to detect viral and atypical bacterial etiologies in all our patients.

From a total of 288 samples analyzed from children under 5 years old with a probable diagnosis of Pertussis, the most common pathogen isolated was ADV in 49% of samples, followed by B. pertussis in 41% from our previous analysis. Although this study was conducted in patients with ARI with a highly suspicious pertussis diagnosis, other studies on children with ARI have identified ADV as one of the most prevalent etiologies Although, the viral and bacterial prevalence may vary widely depending on the population characteristics [8, 33, 34].

Interestingly, our population were children with a highly suspicious clinical diagnosis of pertussis and despite that whooping was present in 46.5% of patients, ADV was the most common etiology isolated. Furthermore, a great number of patients with ADV infection presented with clinical symptoms very common among patients with pertussis such as whooping (51.1%), shortness of breath (43.4%) and vomiting (47.5%). Similarly, a recent study has reported that gastrointestinal symptoms and difficulty breathing are among the most common type of presentations in children. Additionally, ADV has been historically identified as a major cause of pertussis-like syndrome, which results in the likelihood of a pertussis misdiagnosis in the absence of laboratory confirmation [36–38]. In this way, we have shown that patients with infection by ADV and B.pertussis as a single infectious agent have similar symptoms (Fig. 1).

It is also important to highlight the presence of Mycoplasma pneumoniae (26%) and Chlamydia pneumoniae (17.7%) among our patients. In a previous study, in children with ARIs, we reported a very similar prevalence of Mycoplasma pneumoniae and Chlamydia pneumoniae in 25.2 and 10.5%, respectively. Demonstrating the high prevalence of these atypical bacteria among Peruvian children with ARIs. Our results from this current study also make noteworthy that clinical manifestations by Mycoplasma pneumoniae and Flu-B, ADV, or B. pertussis are distinguishable when the infection is due to infectious agent alone. However, in coinfections the symptoms were undestinguibles (see Table S4).

Additionally, in our previous study coinfections between these bacteria and viruses were also frequent; present in 67.4% of samples, coinfections between were the most common combination and the association between Mycoplasma pneumoniae with VRS-A was the most frequent one observed in 9.59% of patients. Surprisingly, in this study, we have observed 58% of coinfections in our samples, again being the viral-bacterial association the most frequent and the most commonly detected coinfection involving Bordetella pertussis-ADV and Mycoplasma pneumoniae-ADV with frequencies of 12.2 and 6.5%, respectively. Another study in children, although in patients with community-acquired Pneumonia, also have reported coinfections in M. pneumoniae as the most common bacteria detected in association with a virus. Thus, to avoid under-diagnosis, pertussis should be considered in patients with cough, especially if chronic, even when M. pneumoniae have been documented.

Another common coinfection was B. pertussis and Flu-B present in 9 patients. Although viral-bacterial coinfections are commonly associated with worse clinical courses and longer hospitalizations [17, 19, 20]. Recent investigations have reported similar clinical outcomes in infants hospitalized with B. pertussis and another respiratory virus coinfection. However, noteworthy attention should be given to the B. pertussis and ADV coinfection in infants. A study compared infants with RSV and RSV-B. pertussis coinfection reporting similar disease severity; however, patients with this coinfection clearly needed more respiratory care and nutritional support. Consequently, our only patient with RSV-A and pertussis presented with cyanosis and required advance respiratory support.

The variations in the rate and pattern of coinfection in patients with ARIs may be related to seasonal and geographical factors. In our study, we intended to describe all detected pathogens and their seasonal distribution. Even though we were not able to describe any clear pattern, it is worth mentioning the high prevalence of ADV and M. pneumoniae across all of the study period, as well as the increasing prevalence of B. Pertussis on 2012.

There are sixteen recognized serological subtypes of type A influenza virus hemagglutinin (H1 through H16) and 9 type A neuraminidase subtypes (N1 through N9). Among the combinatorial diversity of 144 possible A/HN subtypes, relatively few subtypes have been identified as causes of human disease. Four pandemic outbreaks in the last century, one catastrophic, appear to have introduced the subsequently prevalent seasonal human influenza virus subtypes A/H1N1 (Spanish flu, 1918), A/H2N2 (Asian flu, 1957), A/H3N2 (Hong Kong flu, 1968), and A/H1N1 again (Swine flu, 1976; Russian flu, 1977). The current year 2009 has been marked by a late season pandemic-scale emergence of a novel A/H1N1 outbreak strain, raising immediate concerns for public health as well as for pork and poultry production industries worldwide.

As with the few common subtypes of human type A influenza viruses, there are similarly few subtypes of type A influenza viruses that are associated with most influenza infections of swine, horses or dogs. In distinct contrast, wildfowl species are natural hosts and a global reservoir for the majority of possible influenza A/HN subtypes. Many of these variant strains appear to be associated with endemic infections, often asymptomatic in avian hosts. Incidental infections of humans by avian influenza viruses have been documented for avian influenza subtypes A/H5N1, A/H7N2, A/H7N3, A/H7N7, A/H9N2, A/H10N7 and A/H11N9. Recent outbreaks of “bird flu” may foreshadow an eventual pandemic outbreak, in the emergence of strains and variants with enhanced pathogenicity, virulence and transmissibility in human hosts. Examples of such outbreaks include A/H5N1 Hong Kong, 1997; H9N2 Hong Kong, 1999; A/H7N7 Netherlands, 2003; A/H5N1 Southeast Asia, 2004. Some avian A/H5 and A/H7 strains of influenza virus are recognized as highly pathogenic (HP) in domestic poultry and concerns arise that this phenotype may carry over to infections of humans. Since 1997, human infections associated with the Eurasian-African lineage of A/H5N1 HP avian influenza virus have been associated with 467 documented cases in 15 countries with high mortality (282 deaths) [2; updated 30 December 2009].

Fortunately, infectious transmission of such avian influenza virus strains between humans continues to be limited. However, history suggests that further evolution of these or other type A influenza strains could emerge as a next pandemic strain. Similarly, variant type A influenza virus strains have emerged from time to time, imposing serious costs and burdens upon poultry and livestock production.

Because the natural history and the molecular biology of influenza viruses reflect such viral genome diversity, there is a critical need for rapid, sensitive, specific, and informative assays to detect and characterize any subtype of influenza virus. Benchmark standard methods that employ propagation of virus in cell culture or in embryonating chicken eggs, with assays using panels of specific serological reagents, or reverse transcriptase polymerase chain reaction (RT-PCR)-based assays, using panels of short oligonucleotide primers and probes, are either slow and time consuming, or expensive. As prevailing strains of avian influenza continue to evolve and diverge, diagnostic assays that are based only on specific recognition of short signature sequences or peptide biomarker loci will increasingly fail, through false-positive and/or false-negative results. This will adversely impact critical decision-making.

This report describes a re-sequencing pathogen microarray (RPM)-based assay for simultaneous detection, identification and characterization of any subtype of type A human or avian influenza virus, based on rapid, sensitive and specimen-specific determination of nucleotide sequences from viral hemagglutinin, neuraminidase, and other genes.

Influenza viruses are major human pathogens that cause a significant number of illnesses and deaths each year during the seasonal epidemic. In the United States alone, seasonal epidemic influenza has been estimated to result in 31 million outpatient visits and an annual total economic burden of $87 billion (1, 2). The elderly, children, and individuals with underlying medical conditions are at risk of increased morbidity and mortality caused by influenza virus infection (3, 4). Furthermore, emerging influenza virus strains have the potential of sustaining efficient human-to-human transmission and causing a global pandemic, such as the recent outbreak of influenza A(H1N1)pdm09 (5). In terms of treatment options, neuraminidase inhibitors (NIs) are currently the first-line influenza virus antiviral drugs. NI administration is recommended during the early phase of the disease (between 24 and 72 h after illness onset), when the replication of influenza virus peaks (6). At a community level, timely implementation of patient isolation and social distancing measures, including school closures, have been shown to reduce viral transmission (7, 8). The control of influenza virus infection highlights the critical role of an efficient diagnostic assay that enables prompt identification of an infected person and the initiation of subsequent disease management.

At present, there is a wide range of techniques available to achieve different levels of diagnosis, including serology, conventional virus culture, direct fluorescent antibody (DFA) test, rapid immunoassay (rapid test), and nucleic acid test (NAT). Serology and virus culture involve lengthy laboratory processes (2 to 14 days) that render these methods unsuitable for rapid clinical diagnosis (9, 10). R-mix cultures have been widely used for the detection of influenza viruses, with an improved turnaround time of 1 to 2 days (11, 12). DFA, although it significantly reduces the turnaround time to 2 to 6 h, is generally less sensitive than virus culture and requires trained personnel to perform and interpret results (13, 14). Rapid tests for influenza virus based on viral antigen detection by lateral flow immunochromatographic dipstick assay have become widely used in point-of-care settings, such as emergency rooms, doctors' clinics, etc. Rapid tests are user-friendly and can be performed with minimal training without the requirement of complex equipment. In terms of turnaround time, rapid tests confer the highest efficiency, with the results known within 10 to 15 min. However, it has been shown that rapid tests exhibit inconsistent test performance, especially in the detection of A(H1N1)pdm09 viruses (15–18). Due to the limitations of the rapid immunoassays and the increasing availability of molecular technologies, a number of molecular assays for in-house research and diagnostic use have become available (14, 19). The majority of the molecular tests are based on real-time quantitative reverse transcription-PCR (qRT-PCR) and require trained operators and specialized equipment for the extraction and amplification steps (20–23). In addition, the cost and unavailability of centralized testing pose a significant threat to the control of influenza virus in resource-limited settings.

Here we describe a new molecular assay for the detection influenza A and B virus nucleic acids that was developed on the SAMBA (simple amplification-based assay) molecular platform to achieve a high level of performance while providing a fast and easy-to-use diagnostic solution (24). The test, the SAMBA Flu duplex test, couples isothermal amplification with visual detection of nucleic acid on the dipstick to allow a simple and sensitive diagnosis of influenza virus A and B infections.

The University Hospital of Alexandroupolis is a report center of the H1N1 virus for the region of Thrace in Greece. The hospital consists of more than fifty departments, one of which is the Unit of Infectious Diseases. During the influenza epidemic a special department for flu was established in which all patients with flu symptoms and/or signs were referred. Patients with positive flu test were transferred in an 8 bed unit with negative pressure especially designed to quarantine and isolate patients with airborne transmitted viral infections.

From 10th August until 31st December 2009, 33 cases of confirmed H1N1 influenza A virus were hospitalized and quarantined in the Unit of Infectious Diseases. All patients with flu-like symptoms (sore throat, cough, rhino rhea, or nasal congestion) and fever >37.5°C were admitted in the Unit of Infectious diseases and gave pharyngeal or nasopharyngeal swabs. The swabs were tested with real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) as in previous reported studies. It should also be mentioned that although RT-PCR is the most sensitive and specific test for the diagnosis of influenza virus infection, upper respiratory tract specimens are not as specific (~80%) as lower respiratory tract specimens (~100%). All results were given in a period of time from 8 to 48 hours, and all patients remained under quarantine and isolation in negative pressure chambers according to WHO guidelines. Our department prefers to use the Pneumonia Severity Index in order to evaluate the severity of the disease. However, this score was not different between the two groups. The Pneumonia Score Index was calculated for patients in both groups and the Class range was between II-IV. We repeated HINI test 7 days after admission and no patient negative in the initial test became positive.

In total, 60 patients were admitted in a four month period, of whom 33 were H1N1 positive (group A) and 27 negative (group B). The 33 H1N1 positive patients remained under quarantine and isolation, while the 27 negative patients were moved to the Department of Internal Medicine. Patients were monitored until discharge, with symptoms and signs recorded daily. Return to normal body temperature was defined as a temperature of less than 37°C for 1 day after withdrawal of antipyretic treatment. The criteria for discharge were absence of hypoxemia, normal chest x-ray and temperature <37°C for 1 day without antipyretic treatment.

Upon admission procalcitonin (PCT) evaluation was performed in 51 patients (25 group A/26 group B), for nine patients this exam was not available due to lack in reagents in our laboratory. Also urine antigen for Legionella and Streptococcus pneumoniae was tested upon admission in all patients but they were negative. Upon admission sputum stain was given in 12/33 patients in group A and 15/27 in group B. The rest of the patients did not produce enough sputum quantity for staining or did not cooperate in giving sputum. Finally blood cultures were collected in 49/60 patients when the body temperature exceeded 38°C, but no results came positive.

We described a case series of sixty patients who were hospitalized in the Unit of Infectious diseases from 10th August to 31st December 2009 with flu-like symptoms and were tested with RT-PCR for H1N1 virus. Of these, 33 patients were positive for H1N1, while the remaining 27 were negative. The main differences between these two groups and corresponding clinical messages are summarized underneath.

In this case control study we included all patients with influenza symptoms admitted to the emergency flu department according to their attendance. Limitations of the study include that our data represent the experience of a single center, that procalcitonin test was given only in 51/60 patients and also Erythrocyte Sedimentation Rate (ESR) was sporadically collected during the follow up of the patients and so was not evaluated. Bacterial pneumoniae in association with influenza has been considered a important factor leading to poor patients outcomes in prior pandemics. Even though none of the blood cultures were positive, we were unable to evaluate the effect of bacterial co-infection on patient outcomes, since blood cultures were obtained in only 17% of the study population (when fever ≥38°C) and workup for atypical pathogens was not performed. Although bacterial co-infection was not documented, the majority of the study population was treated with antibiotics. Prior publications failed to demonstrate any significant involvement of bacterial pathogens in hospitalized patients with 2009 H1N1 virus pneumonia. During the initial evaluation in 4/27 patients of group B and 6/33 of group A an antibiotic treatment was prescribed by a General Practitioner and none of these patients had a sputum culture at that time. Furthermore, we did not receive cultivable sputum samples from all patients. We supposed that false negative culture in pneumonia patients is mainly due to mixed microbial flora or the natural colonization admixture of the upper airway. The subgroup of patients with pneumonia in both groups is so small that any statistical analysis is impossible and the power of the sample is quite small. Future studies are necessary to define the best treatment of 2009 H1N1 virus pneumonia and the role of combination antiviral therapy.

The lack of significant differences in the percentages of patients with hypoxemia between the two groups is probably due to the proximal number of patients with local patchy shadowing observed in group B and group A. (Table 2.)

Obesity is known to be associated with influenza A (H1N1) viral infection, but in this cohort we observed that in group B there was a larger number of obese patients in opposition to group A (88.8% vs. 54.5%) (p = 0.009). We were unable to explain the reason that the majority of H1N1 patients were not obese in our study as in previous reported studies. Obesity is not a risk factor for poor outcomes in patients with seasonal influenza, but obesity has been suggested as a risk factor for poor outcomes in patients with 2009 H1N1 influenza infection in the USA.

In our case control study a large number patients suffering from lymphoma were observed, because these patients received chemotherapy regimen making them vulnerable to respiratory infections. Patients in group B had elevated C-reactive protein (mean 12.8 vs. 5.74) and white blood count WBC in comparison to group A (mean 10.528 vs. 7.114) suggesting a microbial infection already upon admission. These elevated values (C-reactive protein and WBC) are known to be associated with bacterial infection and early antibiotic treatment prevents progression of the disease as reported in previous studies.

Symptoms from oseltamivir were mainly observed in group A (nausea 4.5% vs. 1.5%, diarrhea 4.5% vs. 1.5%, vomiting 1.4 vs. 0.5%) probably because of the larger dose and prolonged treatment with oseltamivir (5.8 vs. 1.93) as previously reported. However, it was difficult to distinguish the pharmaceutical side effects of osetalmivir (tamiflu) from influenza symptoms in patients receiving antiviral treatment for less than 5 days. Oseltamivir should be given until proof of negative RT-PCR result, since if a patient is positive, it prevents progression of the disease as shown in previous observational studies.

Moreover, mean duration of hospital stay was 5.85 in group B vs. 6.11 days in group A, because of the time needed for normalization of chest radiographs. Nevertheless, there were no significant differences between the two groups and the days of hospitalization were limited due to early oseltamivir for group A and antibiotic treatment for group B as previously explained.

Lastly the mean young age of the patients in both groups, and the small number of co-morbidities observed in our sample of patients, possibly were also responsible for having overall mild clinical course.

A total of 288 patients under 5 years old with a probable diagnosis of B. ertussis were studied thouroughly for specific etiological identification. More than 80% of our study population were infants between 1 to 5 months old with a slightly higher number of males (56.3%). The group of infants between 29 days – 2 months-old (27.4%) and the group between 3 and 5 months-old (27.4%) were the most predominant, closely followed by the group between 2 and 3 months-old (26.4%) (Additional file 1: Table S1).

From our previous study, 118 cases of Bordetella pertussis were confirmed via PCR, leaving potentially 59% of samples without etiological identification. Thus, all 288 were analyzed for the presence of Influenza-A (Flu-A), Influenza-B (Flu-B), RSV-A, RSV-B, Adenovirus (ADV), Parainfluenza 1 virus (PIV-1), Parainfluenza 2 virus (PIV-2), Parainfluenza 3 virus (PIV-3), Mycoplasma pneumoniae and Chlamydia pneumoniae. The most common pathogen isolated was Adenovirus at 49% (141/288), followed by Bordetella pertussis at 41% (118/288), Mycoplasma pneumonia at 26% (75/288) and Flu-B at 19.8% (57/288) (Additional file 1: Table S1-A). The identification of these infectious agents has made it possible to establish remarkably that coinfections were present at 58% (167/288) of patients. Thus, cases of infection due to a single infectious agent were 28.8% (80/288), and where the presence of ADV was 10.2% (25/80) and for B. pertussis was 9.8% (24/80), followed by M. pneumoniae with 6.1% (15/80). Furthermore, the prevalence of these infectious agents were accumulated in children under 5 months of age (Additional file 1: Table S1-B).

As indicated above, in infected children (247 cases) coinfections stand out considerably (Additional file 2: Table S2). The coinfections found involve 2 to 6 different infectious agents, being the most frequent the coinfections of 2 agents with 39.6% (97/247) and those involving 3 agents with frequency of 23.2% (57/247). The most frequent association was the bacterial-viral coinfection, and the combination between Bordetella-ADV and Mycoplasma-ADV were the most common involvement reported with 12.2% (30/247) y 6.5% (16/247), respectively. However, the Bordetella-Mycoplasma association was very reduced (Additional file 2: Table S2), In addition, it is interesting to note that the associations in coinfections increase the frequency of infectious agents such as Chlamydia, Flu-B and RSV-A as observed in Additional file 1: Table S1 (compare 1A with 1B).

Regarding vaccination status, 65.25% (77/118) of the positive cases were unvaccinated. However, the majority of these children (40/77) were under two months of age. An unknown vaccinated status was observed in 5.93% (7/118) of patients positive for B. pertussis. A marked decrease was observed in children who had received at least one dose of vaccination, with a prevalence of 18.64% (22/118) (Additional file 3: Table S3).

In our population, the most common clinical symptoms registered at admission were vomiting (47.2%), whooping (46.5%) and shortness of breath (43.1%), followed by fever (35.4%) and cyanosis (28.8%). A wide spread distribution of symptoms distribution was observed when patients symptoms were individually assessed based on etiological group. Only 6 pathogens had symptoms that were present in more than 50% of each group. For example, vomiting was more commonly reported among children with Flu-A, RSV-A, Parainfluenza-1 and B. pertussis (Additional file 4: Table S4-A). However, the difficulty in establishing clear clinical symptoms associated with infectious causal agents is due to the high frequency of coinfections. Therefore, Additional file 4: Table S4-B has recorded the clinical symptoms of cases of infection with a single agent, and the association of these clinical symptoms are shown in Fig. 2. Thus, a clear non-association can be observed between Mycoplasma and Flu-B, ADV or Bordetella; the same happens for Flu-B and Bordetella or ADV. This non-association means that the only infectious agent could be identified taking into account the clinical symptoms of the children as shown in Additional file 4: Table S4-B.

The most common complications were acute bronchial obstructive syndrome (ABOS) and pneumonia in 56.6 and 28.1% of our population respectively. ABOS was the most frequent complications among patients with positive samples for RSV-A, Flu-A, ADV, M. pneumoniae, C. pneumoniae and B. pertussis. (Additional file 5: Table S5-A). However, when the complications of children affected by a single infectious agent are analyzed, it is clearly demonstrated that ABOS is also a complication of Flu-B. In addition, it is noteworthy that ABOS occurs in 61% (25/41) of the negative cases (Additional file 5: Table S5-B).

Finally, a seasonal distribution was described for each specific microorganism. Positive samples for ADV and Mycoplasma pneumoniae were observed across the whole study period. On the contrary, most of the B. pertussis cases were detected from May 2011 to March 2012. RSV-A and Chlamydia were mostly detected from March to May 2010; however, the same distribution was not observed in the following year (Fig. 3).

Clinical specimens collected from two sites (HPA Cambridge and WIV-ISP) were screened independently by qualified biomedical scientists for the presence of influenza virus according to their routine testing protocols.

This study reported a high prevalence of respiratory viruses in children ≤ 5 years using a custom assay and an FTD assay. Good concordance was observed for all the viruses between both assays except for RSVA/B. However larger numbers of positive samples need to be tested for thorough evaluation of less prevalent viruses. The custom primer and probe mix was much more economical than the commercial FTD kit. Our study suggests that this custom multiplex real-time RT-PCR can be used for simultaneous and rapid detection of multiple viruses in resource limited settings. This will help prevent unnecessary use of antibiotics and permit timely initiation of supportive therapy/antiviral drugs if available.

Clinical outcomes were compared among 123 children aged < 3 years with single virus infections and clinical data available (Table 1, Table 2). A total of 21 children were previously excluded from comparative analyses due to incomplete documentation or hospital transfer without follow-up data. Excluded cases were equally distributed among virus groups (Table 1). High levels of CRP (≥ 35 mg/l) or WBC (≥ 15 x109/L) and antibiotic treatment were equally distributed among virus groups and suggested no differences in potential serious bacterial infections. Bacterial cultures and PCRs were often negative and were equally distributed among individual virus infections. This supports the assumption that there were no differences in potential serious bacterial infections among virus groups. Hospital admission (~80%) was equally distributed and fortunately there was no mortality. Overall and individual comparisons were made among children with RSV, HRV, FLU and Other single virus infections (Table 2) following multivariate adjustment for possible confounders.