2017.

The disease was suspected to be of "unknown origin pneumonia" at the beginning of the outbreak; and quickly SARS and/or avian influenza were precluded with SARS-coronavirus and H5N1 specific detection, bacterium infection was precluded as well. The first case was reported on 15, cases accumulated in a short period and peaked on 17 Jan 2009 (Figure 1). Case epidemiology proceeded for 70 patients (32 reported using the internet reporting system directly and 38 during an active investigation from the four hospitals in Hanzhong). The age of the patients was from 40 days to 9 years; primarily in the 0-3 year range. Endemic distribution was scattered in some villages with the most in Xixiang County without a central tendency. Among the 70 patients, the admitting diagnosis was 56 with bronchopneumonia, 11 with bronchitis, two with acute tonsillitis, and one with lobar pneumonia. Clinical manifestations included fever (84.5%) with the highest at 40.5°C and a median of 38.8°C; and most cases presented with cough and some with asthma.

Wide range of viruses is known to be associated with respiratory disease in humans. Adenoviruses, coronaviruses, human enteroviruses (HEV), human rhinoviruses (HRV), influenza viruses, parainfluenza viruses (PIV), and respiratory syncytial viruses (RSV) are well-known causes of acute respiratory tract infections (ARTI) in both industrialized and developing countries. Over the last decade, modern molecular techniques have led to the discovery of several previously unknown respiratory tract viruses, including human metapneumovirus (hMPV), two new human coronavirus types [2, 3], human bocavirus (HBoV), and two new human polyomaviruses [5, 6]. The significance of these novel viruses has been reviewed recently [7, 8].

It is widely accepted that common cold is almost always caused by viruses, most frequently by HRV, and viral infections are considered to contribute to the generation of complications of common cold, such as acute otitis media and sinusitis. Moreover, different viruses, including influenza viruses and RSV, are also frequently detected in samples obtained from patients with lower respiratory tract infection (LRTI), either alone or together with pathogenic bacteria. Several recent reports, including some from Africa, suggest viruses as potential etiologic agents in pneumonia in children [10–13], or exacerbations of asthma [14–16].

Several studies underscore the importance of respiratory tract viruses in Nigerian patients, but these studies were carried out before the introduction of modern molecular diagnostic techniques [14, 17–19]. The present study was designed to identify viral agents associated with respiratory infections among young children in Nigeria using modern, validated molecular techniques. We wanted to explore the presence of different virus groups, including some of the newer ones detected by only molecular techniques.

PCR or RT-PCR was performed with five pharynx swabs specimens collected from the first reported pneumonia patients using primer sets specific for respiratory viruses with the Seeplex RV Detection Set I. The results showed all of the five specimens were positive for human adenovirus and included a further 16 pharynx swabs for a total of twenty-one pharynx swabs: 14 were positive for adenovirus, three had a co-infection with respiratory syncytial virus, two positive for rhinovirus, one positive for parainfluenza 3, and four were negative. Of the 14 adenovirus positive samples, partial hexon gene sequencing results showed the 12 specimens were species B of HAdV, nine HAdV-7 and three HAdV-3 and amplification bands of two specimens were too weak to perform sequencing.

To determine viral causes of influenza-like illness in Uganda.

All respiratory viruses may cause symptoms such as nasal congestion, runny nose, wheezing, and cough. We found no significant association between the viruses and a specific symptom.

Viral aetiology, prevalence and diversity data in people with influenza like illness (ILI) and/or acute respiratory illness (ARI) in Africa, (especially in West Africa), are scarce and often limited to the influenza viruses’ infection. Following the last influenza pandemic episode, few global and pediatric studies were conducted in some countries of the sub-region, and only a limited number of studies have described the etiology of ILI due to viruses including non-influenza respiratory virus. However, no study has been conducted to describe the prevalence and the diversity of respiratory viruses (influenza and others) in West African elderly people. In studies done elsewhere, it is well established that older people, when compared with younger adults, are at greater risk of significant morbidity and mortality from complications arising from influenza. For example in the United States alone, up to 40% of non-pneumonic lower respiratory illnesses in the elderly have been associated with respiratory viral infection, and an estimated 54,000 deaths annually have been attributed to the influenza and respiratory syncytial viruses (RSV).

It should be highlighted that in Senegal the number of elderly people in consultation in healthcare centers for influenza like illness (ILI) is very low. Indeed, routine influenza monitoring in Senegal showed that samples from people above 50 years old represent only 3.7% of the total, over a 16 year surveillance period. Some practices such as auto-medication and the use of traditional medicine to treat ILI largely explain this situation with the socio-economic situation being another contributing factor.

Thus the main aim of this study was to determine the prevalence and the diversity of respiratory viruses associated with ILI cases in adults over 50 years old.

Children with viral infection in the upper respiratory tract showed symptoms such as runny nose, cough, and hoarseness. Some of them also present lower respiratory tract symptoms such as wheezing, severe cough, breathlessness, and respiratory distress, which may be due to bronchiolitis or pneumonia.

We divided the patients according to three clinical manifestations: pneumonia, bronchiolitis, and bronchitis, and investigated whether the detected viruses were associated with a specific clinical manifestation. Our analysis showed that in most cases, RSV infections induced bronchiolitis (n = 45), followed by pneumonia (n = 22, p=0.004) and bronchitis (n = 6, p=0.0015). EV/Rhi infections more often induced pneumonia or bronchiolitis (n = 19 for both) instead of bronchitis (n = 6, p=0.03) (Figure 2). Other viruses showed a similar prevalence of each clinical manifestation.

Human influenza virus infection replicates primarily in the respiratory epithelium. Other cell types, including many immune cells, can be infected by the virus and will initiate viral protein production. However, viral replication efficiency varies among cell types, and, in humans, the respiratory epithelium is the only site where the hemagglutinin (HA) molecule is effectively cleaved, generating infectious virus particles. Virus transmission occurs when a susceptible individual comes into contact with aerosols or respiratory fomites from an infected individual.

The ferret has traditionally been used as a model of influenza transmission as most human influenza viruses do not need any adaptation to infect and transmit among ferrets. Studies in ferrets have identified the soft palate as a major source of influenza viruses that are transmitted between individuals. Notably, the soft palate is enriched in α2,6-linked sialic acids, which are preferred by the hemagglutinin proteins currently found in circulating human influenza viruses. This enrichment also occurs in the soft palate of humans.

The primary mechanism of influenza pathophysiology is a result of lung inflammation and compromise caused by direct viral infection of the respiratory epithelium, combined with the effects of lung inflammation caused by immune responses recruited to handle the spreading virus (Table 1). This inflammation can spread systemically and manifest as a multiorgan failure, but these consequences are generally downstream of lung compromise and severe respiratory distress. Some associations have also been observed between influenza virus infection and cardiac sequelae, including increased risk of myocardial disease in the weeks following influenza virus infection. The mechanisms of this, beyond a general inflammatory profile, are still unresolved [5, 6].

Severe acute respiratory infections (SARI) are one of the major causes of illness and death worldwide and are the third most common cause of death among children. Acute respiratory infections (ARI) cause more deaths in children < 5 years with most cases reported from India (43 million), China (21 million), Pakistan (10 million), Bangladesh, Indonesia and Nigeria (56 million). Respiratory infections can be caused by many viruses, both DNA and RNA. These include the Respiratory Syncytial Virus (RSV), human Parainfluenza Virus (HPIV), Influenza A Virus (Flu A), Influenza B Virus (Flu B), human Adenovirus (HAdV), human Coronavirus (HCoV), human Rhinovirus (HRV), human Metapneumovirus (HMPV) and human Bocavirus (HBoV). A new wave of viral diagnosis was established with the development of Polymerase Chain Reaction (PCR) techniques in the 1990s. PCR is more sensitive and rapid than conventional methods for detection of respiratory viruses. Different respiratory viruses present with similar signs and symptoms and can’t be differentiated symptomatically or clinically. Tests capable of rapid simultaneous identification of various viruses at the same time can help expedite initiation of appropriate therapy. Uniplex RT-PCR requires individual amplification of each virus under study which is expensive, time consuming and laborious. To overcome this, multiplex real-time PCRs targeting the detection of multiple pathogens simultaneously have been developed commercially but they are very expensive. There is a need to develop cheaper systems for rapid simultaneous identification of various viruses. The present study compares custom real-time multiplex PCR primers and probes for the simultaneous detection of 18 respiratory viruses with an in-vitro diagnostics (IVD) approved fast track diagnostics (FTD) kit.

Samples were not collected from patients with chronic respiratory ailments; non-consenting caregivers, with history of hospitalization in the preceding 14 days, not admitted in hospital and children aged > 5 years.

The respiratory syncytial virus (RSV) has great importance as a causative agent of nosocomial infections, especially for its particles being highly contagious.1

Respiratory syncytial virus severe clinical outcomes in children and immunocompromised patients are largely known; however, the studies including asymptomatic individuals with different levels of exposure to this pathogen in different sets are scarce.2, 3Asymptomatic respiratory viral infections occur in a variable frequency, and among young children, it can be high.4 The occurrence of transmission between children and their asymptomatic contacts is still insufficiently studied, despite their importance in the virus transmission chain. It is known that asymptomatic excretion of RSV occurs in 15%‐20% of the infected healthcare workers (HCWs).1 In other adults, the asymptomatic RSV infection is still barely studied and the researchers keep trying to understand the detection of genetic particles in their samples.5 For example, a few studies have been conducted regarding the frequency of RSV infection in patients HIV positive.6, 7

This study aims to evaluate the occurrence of symptomatic and asymptomatic infections caused by RSV by analyzing samples collected from patients, healthcare workers and companions of patients in a university hospital complex. The frequency of RSV detection in individuals with different risk factors for acquiring infection is described. Comparisons of viral load were also performed. The individuals included in this study were HCWs, HIV‐infected patients, children, and caregivers of children with respiratory symptoms.

The role of respiratory infections as key causes of morbidity and mortality among military trainees is well-recognized. Compared to the young healthy adults’ population, military trainees undergoing basic military training course appear to be at increased risk of acquiring and transmitting respiratory infections (1). Every year, a large number of cases of respiratory infections occur in basic military trainees worldwide which the majority of them are resulting from viral infections. Respiratory infections have a tremendous impact on the military population and are responsible for 25–30% of hospitalization in the U.S. military (2). Affected trainees generally require a sufficient period of recovery which can lead to a longer training duration and a significant higher rate of viral transmission to newer cohorts (3). The higher vulnerability to respiratory disease epidemics observed in military trainees has been attributed to several causes including crowded habitation, demanding physical training program, stressful working environment, and insomnia (4, 5).

Numerous previous studies reported that adenoviruses, influenza A and B, human rhinoviruses, and coronaviruses are the predominant viruses detected in the military population. Outbreaks of adenovirus-associated respiratory disease have been reported globally in the military environments (3, 5-8). It has been suggested that adenovirus infection is associated with male gender, as well as direct contact with an infected person with respiratory symptoms 10 days prior to the onset of illness (8). Outbreaks of influenza viruses A and B have also been associated with much morbidity and mortality, especially influenza A (H1N1) pdm09 virus infection (9-13). It has been proposed that crowded living quarters, obesity, asthma, and age group (younger than 40 years) are amongst the major risk factors for acquiring influenza infections (14). Human rhinoviruses are the most important causative agents of the common cold and associated with more complicated upper respiratory tract infections (15,16). Approximately, all the human rhinoviruses have been detected in military trainees during respiratory infection (17). It has been also well documented that human rhinoviruses are associated with lower respiratory tract infections (18, 19). Nevertheless, there are other viruses associated with respiratory infections which are not well studied in military populations, including respiratory syncytial viruses (20-22), human bocaviruses (23-25), human parainfluenza viruses (26), metapneumoviruses (27), and echoviruses (28).

Until now, there is no study which aimed to investigate the prevalence of viral agents responsible for respiratory infections among the military population in Iran. Using micro-array technology, the objective of our study was to evaluate the molecular epidemiology of 17 viral pathogens causing respiratory infections among 400 military trainees in a large military training camp in Tehran, over a period of time from January to March 2017. The data resulted from this study can be employed to take preventive measures aimed to reduce the disease burden and prevent future outbreaks.

Lower respiratory tract infection (LRTI) remains one of the major causes of mortality and morbidity in children under five years globally. Viruses have already been recognized as important etiologies of respiratory infections with influenza virus which is considered as the main contributor. The epidemiology and public health impact of influenza infections are relatively well described as many studies and surveillance have been conducted in part of pandemic preparedness [2–5]. Most of the countries in the world, including Indonesia, have developed influenza surveillance, influenza-like illness (ILI) surveillance, and severe acute respiratory illness (SARI), which form the network under WHO through Global Influenza Surveillance and Response Systems (GISRS) [4–8]. This network improves influenza disease control by providing support on influenza vaccine recommendation, laboratory diagnostic tools, antiviral, and public health risk assessment. As influenza virus contributed only less than 30 percent of viral respiratory infections, there is an urge to investigate the contribution of other respiratory viruses for improving respiratory disease control program.

Recent advancement of molecular technology supports the investigation and characterization of several respiratory viruses. The molecular technology improves the capability to study respiratory viruses, which are previously identified: rhinovirus, adenovirus, respiratory syncytial viruses, parainfluenza virus, and also the new emerging viruses/strain viruses: MERS coronavirus, human metapneumovirus, and human rhinovirus strain C. Multiple detection platforms, which recently have been developed, allows relatively inexpensive and timely detection of several viruses [9–11]. The detection of multiple respiratory viruses will accommodate the efforts to determine the epidemiology of noninfluenza respiratory viruses in the community, which will further help the respiratory disease control program including the use of antimicrobial agents.

Previous results of the investigation on acute respiratory infection patients in several countries showed the difference in the prevalence of respiratory viruses among studies [3, 13–15]. Study design including the case definition, study population, time of the study, and diagnostic tools being used have been considered as factors that influenced the variation. Each virus has different seasonality circulation and an age-related prevalence that can lead to a specific pattern of virus cocirculation in many studies [17–19]. Moreover, the occurrence of virus coinfection in which two or more viruses are detected in a single patient has been described in recent studies using multiple pathogen detection platforms [20–22].

There are limited studies on viral pathogens of respiratory tract infection in low-middle income countries including Indonesia. Previous studies have been conducted mostly focused on specific viral pathogens, especially influenza [6, 23, 24]. The prevalence of noninfluenza respiratory infections is relatively unknown. Therefore, this study has an objective to investigate the prevalence of viral etiologies from ILI cases in Indonesia.

Acute respiratory infections (ARIs) pose a significant public health problem worldwide, causing considerable morbidity and mortality among people of all age groups. Children are on average infected two to three times more frequently than adults.. There are more than 200 respiratory viruses that can cause ARIs. Human respiratory syncytial virus (HRSV), human rhinovirus (HRV), human metapneumovirus (HMPV), human parainfluenza virus (HPIV), human enterovirus (EV), influenza virus (IFV), human coronavirus (HCoV), adenovirus (HAdV), and human bocavirus (HBoV) are the most common viral agents associated with ARIs, accounting for around 70% of ARIs [3, 4]. The frequency of mixed respiratory viral detection varies from 10% to 30% in hospitalized children [5–7]. In addition, several new human respiratory viruses have been described in recent years, including human metapneumovirus [8, 9], human bocavirus, and novel human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), human coronaviruses NL63 (HCoV-NL63), HKU1 (HCoV-HKU1), and Middle East respiratory syndrome coronavirus (MERS—CoV).

Although the majority of ARIs are associated with respiratory viruses, antibiotics are often used in the clinical treatment of ARIs. As children with ARIs often have similar clinical symptoms, studying the clinical hallmarks of children with virus-related ARIs and the spectrum of respiratory viruses will help in developing more accurate treatments for ARIs. Rapid diagnosis is important not only for timely treatment starting but also for the detection of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment [15, 16].

Western Siberia plays a key role in ecology, epizootiology and epidemiology of emerging diseases. This territory was involved in the circulation of A/H5N1 and A/H5N8 avian influenza viruses in 2005–2017 [17, 18]. These viruses were spread by wild birds’ migration. In Western Siberia migratory flyways of birds’ wintering in different regions of the world: South East Asia, Central Asia, Middle East, Hindustan, Europe, and Africa—cross. For this reason, there is high probability of the emergence of humans and animal influenza viruses reassortants, as well as emergence of local outbreaks of human morbidity caused by uncommon variants of influenza viruses. Furthermore, Novosibirsk is the largest transport hub in this part of Russia with numerous international connections, that is important for the spread of ARIs [19, 20].

The prevalence of respiratory viruses among children with ARIs differs in different regions and varies over time [21–25]. Thus, to better understand the epidemiology of Acute Respiratory Infections in Novosibirsk region, we investigated etiology of ARIs in children admitted to Novosibirsk Children’s Municipal Clinical Hospital in 2013–2017.

Acute respiratory infections (ARIs) are common and contribute significantly to morbidity and mortality. They are the leading causes of outpatient visits and hospitalizations in all age groups, especially for children under 5 years of age.1 Most ARIs in children and outpatients are caused by nine common respiratory viruses, including respiratory syncytial virus (RSV), influenza virus A, influenza virus B, rhinovirus, adenovirus, parainfluenza virus, coronavirus, human metapneumovirus, and boca virus2, 3 Additionally, atypical pathogens, such as Mycoplasma pneumoniae, are also major causes of ARIs in children. The symptoms caused by these pathogens are largely similar, thus definitive diagnosis requires effective laboratory testing. By using multiplex assay targeting these pathogens, early diagnosis can be made in a timely manner. Consequential antimicrobial or antiviral therapy may thus be administrated promptly and appropriately.4 Most importantly, the early diagnosis of influenza viruses, which are contagious, is beneficial for early isolation of patients, thus reducing the spread of influenza viruses.

The routine clinical laboratory testing for respiratory viruses is largely conducted by direct fluorescent‐antibody assays and rapid antigen tests in China. Given the poor sensitivity and complicated manual operation, these methods have been gradually replaced by nucleic acid amplification tests (NAATs), which are more sensitive and more specific. However, majority of the NAAT kits are based on real‐time polymerase chain reaction (PCR), which can only detect one or two pathogens of ARIs within a single tube, thus are not syndromic testing.5 The clinical and economic impacts of syndromic testing for respiratory pathogens have been evaluated in several studies. Overall, the implementation of syndromic testing can decrease the time of diagnosis,4 decreased healthcare resource utilization,6 decrease inpatient length of stay and time in isolation,7 and improve antiviral use for influenza virus‐positive patients.8

SureX 13 Respiratory Pathogen Multiplex Kit (ResP) is a syndromic multiplex molecular test for simultaneous detection of 13 pathogens in a single tube. The aim of this study was to evaluate the application of the ResP for detection of respiratory pathogens in outpatients with flu‐like manifestations.

Acute myositis accompanied by rhabdomyolysis may rarely happen, most commonly in children who present with extreme tenderness of lower extremities, and the laboratory investigation shows marked elevation of serum creatinine phosphokinase and myoglobinuria. Myocarditis and pericarditis have also been rarely described in clinical cases, but demonstrated in autopsy studies [83, 84]. Central nervous system complications associated with influenza include encephalitis, acute disseminated encephalomyelitis, transverse myelitis, aseptic meningitis, and Guillain-Barre syndrome [85–87] (Table 2).

Onsite, the aetiology of respiratory infections was identified in 11 of 42 (26%) symptomatic cases. Before the Games, two cases of influenza B were detected by local healthcare services using antigen detection. POCT in the team’s medical room detected one case of influenza A virus, three cases of influenza B virus and five cases of respiratory syncytial virus A. The aetiology of the common cold was identified in six athletes and in five staff members. All six patients with an influenza virus infection were treated with oseltamivir. Oseltamivir prophylaxis was given to their 32 contacts (11 athletes). None of them developed a symptomatic infection.

Although acute respiratory illness is a major cause of morbidity and mortality among children in sub-Saharan Africa, it has received relatively little attention. This is unfortunate, as underlying diseases such as AIDS, malaria and tuberculosis, which are highly prevalent in the region, can worsen such illnesses. The respiratory viruses known to cause acute illness include human respiratory syncytial virus (HRSV), human parainfluenza virus (PIV), human metapneumovirus and influenza viruses. Until recently, the burden of influenza and influenza-like illness in Africa was considered to be negligible, mainly because of the lack of confirmation assays. Reports from Cameroon and Senegal, however, show that influenza viruses are actively circulating and may be causing regular epidemics.

A clear picture of the contribution of each pathogen to acute respiratory illness is needed in order to improve prevention and clinical management and consequently to reduce the burden of disease. The emergence of the novel influenza A/H1N1 of swine origin in Mexico in April 2009 and its rapid spread worldwide, causing a global pandemic, led the health authorities of the Central African Republic (CAR) to collaborate with the World Health Organization in strengthening biological surveillance of acute respiratory illness.

The aim of the study reported here was to determine the circulation of 2009 pandemic influenza A/H1N1 virus (H1N1pdm09) by molecular methods and to identify the causative viruses, the incidence and the clinical features of acute respiratory illness among infants and young children at sentinel sites in Bangui and three rural areas.

All infants and children aged between 0–15 years who attended sentinel sites in Bangui and three rural areas (Figure 1) for influenza-like illness (ILI) or severe acute respiratory illness between January and December 2010 were included in the study (Figure 2A). The World Health Organization definitions were used for ILI (sudden onset of fever of > 38°C and cough or sore throat in the absence of other diagnoses) and severe acute respiratory illness (ILI symptoms and shortness of breath or difficulty in breathing and requiring hospital admission). The study protocol was approved by the National Ethics Committee of the CAR. Individual written informed consent was sought from the parents or guardians of all participants.

Nasopharyngeal samples were collected from 329 infants and children and within 48 hrs at 4°C to the National Influenza Centre by using rayon-budded swabs with virus transport medium pre-impregnated sponge (Virocult, Medical Wire & Equipment, UK).

RNA was extracted with a QIAmp RNA mini kit (Qiagen) according to the manufacturer’s instructions. Influenza A viruses were detected with a previously described assay targeting the conserved matrix gene for universal detection of these viruses, and H1N1pdm09 virus was identified with a specific one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay (designed by the National Influenza Centre of northern France, Institut Pasteur, Paris; primers and probe available upon request at grippe@pasteur.fr). All specimens were also tested for other respiratory viruses in two previously described multiplex semi-nested RT-PCR assays for detecting influenza A and B viruses, HRSV, human metapneumovirus and PIV types 1, 2, 3 and 4. All assays were performed on an ABI 7500 platform (Applied Biosystems, Foster City, California, USA) with the SuperScript III Platinum One-step Quantitative RT-PCR System (Invitrogen, Carlsbad, California, USA). A specimen was considered positive if the signal curve crossed the threshold line within 40 cycles. The assay limit of detection for pandemic H1N1pdm09 influenza virus is of order of magnitude of 10 copies/μL of initial sample. The assay limit of detection for influenza A and B viruses, HRSV, human metapneumovirus and PIV-3 is of order of magnitude of 10 copies/μL. For PIV-4, the assay limit of detection is of order of magnitude of 100 copies copies/μL, and for PIV-1 and −2, 1000 copies/μL. After amplification, the PCR products were purified and sent to GATC Biotech (Konstanz, Germany) for sequencing.

Student’s t test and the Pearson chi-squared test were used to assess intergroup differences. Statistical analyses were performed with EpiInfo software (V 3.5.1 CDC). A test was considered significant when the p value was < 0.05. The newly obtained sequences were analysed and compared with sequences available in GenBank.

Influenza like-illness (ILI) or acute respiratory infections can be caused by several types of respiratory viruses or bacteria in humans. Influenza viruses, Respiratory Syncytial viruses (RSV) and Parainfluenza viruses are identified as major viruses mostly responsible for ILI and pneumonia in several studies. However practitioners cannot diagnose the infection without a biological test confirmation. Unfortunately, these infections causes are identified in less than 50%.

Réunion Island, a French overseas territory with 850,000 inhabitants, is located in the southern hemisphere between Madagascar and Mauritius in the Indian Ocean (Latitude: 21°05.2920 S Longitude: 55°36.4380 E.). The island benefits from a healthcare system similar to mainland France and epidemiological surveillance has been developed by the regional office of the French Institute for Public Health Surveillance (Cire OI), based on the surveillance system of mainland France. Influenza activity generally increases during austral winter, corresponding to summer in Europe. Since 2011, influenza vaccination campaign in Reunion Island starts in April and the vaccine used corresponds to World Health Organization recommendations for the southern hemisphere.

Since 1996, clinical and biological influenza surveillance has been based on a sentinel practitioner’s network. In 2014, this network was composed of 58 general practitioners (GPs) spread over the island and represented around 7% of all Réunion Island GPs. Nasal swabs are randomly collected all along the year and are tested by RT-PCR for influenza viruses. Among these surveillance samples, 40 to 50% are tested positive for influenza A virus, A(H1N1)pdm09 or B virus by the virological laboratory of the University Hospital Center of Réunion. Thus ILI samples tested negative for influenza are of unknown etiology.

Several biological tools allow identifying respiratory pathogens from nasal swab. In recent years, multiplex reverse transcriptase polymerase chain reaction (RT-PCR) has been developed to identify several viruses simultaneously [7–10]. We therefore used this new method to set up a retrospective study using swabs collected by sentinel GPs from 2011 to 2012.

The main objective of our study was to characterize respiratory pathogens responsible for ILI consultations in sentinel GPs in 2011 and 2012. Secondary objectives were to highlight seasonal trends on respiratory pathogens circulation and to describe occurrence of co-infections, especially during the flu season.

The aetiology of the common cold was finally detected in 30 of 42 patients, in 15 of 20 (75%) athletes and in 15 of 22 (68%) staff members. Nine different respiratory viruses were identified. Coronaviruses (229E, NL63 and OC43) were the most commonly detected viruses (table 1). Two participants had two viruses (respiratory syncytial virus A and coronavirus OC43; and coronavirus 229E and coronavirus NL63). One athlete suffered from two different coronavirus (OC43 and 229E) infections 20 days apart. The recorded PCR threshold values mostly indicated low viral loads (table 1, figure 2). In five athletes and nine staff members (p=0.117), the PCR cycle threshold values were <27, indicating high viral load (figure 2). In 8 out of 20 virus-positive cases, a previous or later nasal specimen was virus-negative.

Nasal mucus samples were taken from 34 asymptomatic subjects due to close contact to a symptomatic subject. Eight samples were virus-positive (rhinovirus 3, coronavirus 229E 3, coronavirus NL63 2).

Multiplexed PCR-testing for nasal bacteria identified H. influenzae in seven subjects and S. pneumoniae in two subjects. Co-detection with a respiratory virus was found in three out of nine cases. No cases of atypical bacteria were identified. Clinically no cases of probable bacterial infections were recorded.

Eleven specimens were not available for retesting in the laboratory. All other POCT results were confirmed in the laboratory.

Four athletes competed with mild symptoms of the common cold. One athlete competed 5 days after the onset of an influenza B infection, another competed 5 days after the onset of a respiratory syncytial virus A infection and two athletes competed five days after the onset of a metapneumovirus infection.

Respiratory tract infections lead to mortality and morbidity in children especially during early years. Among children, more than 80% of respiratory infections are associated with different viral infectious agents. Respiratory virus infections are a major public health problem, due to the ease of spread and considerable morbidity and mortality. The association between respiratory tract infections and different viral pathogens has been reported to vary between 40% and 90% [1–5] globally.

Different studies reported the detection of viruses like human respiratory syncytial virus A (RSV A), human respiratory syncytial virus B (RSV B), human adenovirus (AdV), Human metapneumovirus (HMPV), human coronavirus, and human parainfluenza virus (PIV). Children under the age of 5 years were detected with human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), human parainfluenza virus 1 (PIV-1), human parainfluenza virus 2 (PIV-2), human parainfluenza virus 3 (PIV-3), human parainfluenza virus 4 (PIV-4), human rhinovirus (HRV), human enterovirus (HEV), and human bocavirus (HBoV). Coinfections with different multiple viruses were reported in 15% to 61% of patients. [6–9]. Molecular techniques such as multiplex polymerase chain reaction (PCR) are widely used for the detection and identification of respiratory viruses [10–12] and are helpful in the management and treatment of respiratory infections. Diagnosing respiratory viruses by isolation in cell cultures and serology is time consuming, laborious, expensive, and less sensitive in some cases. Molecular techniques provide quick results with high sensitivity and specificity. Multiplex PCR has been reported as a fast and sensitive assay for respiratory infection detection. Anyplex II RV16 (Seegene, Korea) is a multiplex real-time PCR based kit with Tagging Oligonucleotide Cleavage Extension (TOCE) technology. The pitcher and catcher are two novel components used in TOCE assay for unique signal generation in real time. In TOCE assay detection point is moved from the target sequence to the catcher so it provides the predictable melting temperature analysis for catcher duplex. This process offers the multiplex real-time PCR capability to Anyplex II RV16 kit. [14–17].

Respiratory infections are mostly reported in children living in developing countries. The spread of respiratory infections varies between populations and countries, depending on differences in geography, climate, and socioeconomic conditions [18–21]. The central region (Riyadh region) of Saudi Arabia has a dense population of locals and immigrants whose interaction can affect the transmission patterns of different respiratory viruses. Previous studies have reported the prevalence of a small number of respiratory viruses within different regions of Saudi Arabia, and limited information is available on the seasonal distribution of viruses [22–25]. A better understanding of the local epidemiology and risk factors is critical for the prevention and control of respiratory infections.

This study aimed to determine the distribution of 16 different viruses causing respiratory infections in children, by using RV16, and to compare data on demographic characteristics, symptoms, and single infections or coinfections.

A total of 400 Iranian military trainees with clinical diagnostic criteria for respiratory infection were enrolled in the survey. All participants were male, with a mean age of 21.69 ± 4.9 years (range from 18 to 57 years). Most prevalent complaints of patients referred to the military medical clinic center were sore throat (n=302; 75.5%), rhinorrhea (n=253; 63.2%), cough (n=237; 59.2%), fever (n=237; 59.2%), and nasal congestion (n=202; 50.5%) (Fig. 1). Of the 400 samples, 124 (31%) were positive for respiratory viruses. Human rhinovirus (n=29; 7.2%), human respiratory syncytial virus A (n=29; 7.2%), and influenza B virus (n=24; 6%) were the most frequently detected respiratory viruses in our study, followed by bocavirus (n=12; 3%), influenza A H1N1 (n=9; 2.2%), influenza A H3N2 (n=6; 1.5%), human respiratory syncytial virus B (n=6; 1.5%), adenovirus (n=6; 1.5%), and human coronavirus 229E (n=3; 0.7%). Other viruses including influenza C virus, human parainfluenza viruses, metapneumovirus, and echovirus have not been detected in any of the samples (Table 1, Fig. 2). It's worth noting that no co-infections were detected in our study. The most cases of dyspnea (n=5; 62.5%) were in the group of patients with respiratory syncytial viruses A and B, followed by influenza B (n=2; 25%) and coronavirus 229E (n=1; 12.5%). Sore throat and rhinorrhea were the most frequent symptoms in rhinovirus infection. The most cases of myalgia were seen in influenza B (n=5; 62.5%) and influenza A H1N1 (n=3; 37.5%) infections, respectively (Table 1).

Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a newly emerged novel corona virus, is associated with severe acute respiratory infection with high mortality rates. MERS-CoV is an enveloped, single stranded positive sense RNA virus belonging to lineage C betacoronavirus. It was first isolated in the Kingdom of Saudi Arabia (KSA) from a patient with acute pneumonia who subsequently died of renal failure in September 2012. Although MERS-CoV was suspected primarily as zoonotic in origin butanimal to human transmission is not fully understood yet. Human-to-human transmission has been documented in several clusters among health-care providers and contacts [5–8]. Since 2012, the World Health Organization (WHO) reported 1841 laboratory-confirmed MERS-CoV cases including 652 deaths from 28 countries[9]. All reported cases had an epidemiological link either directly or indirectly and around 80% of the cases were reported from KSA.The crude case fatality rate was 35% and males more than 60 years of age having underlying co-morbid condition are at higher risk of developing life threatening severe disease following MERS-CoV infection[9,12].

Millions of pilgrims from more than 180 countries across the globe unite in the holiest sites of KSA during the Hajj pilgrimage. Elderly pilgrims with different underlying medical conditions and socioeconomic backgrounds from different parts of the world come in close contact during religious rituals in a closely defined area, most likely increasing susceptibility to respiratory tract infection[12,14,15]. More than 30% of pilgrims from Bangladesh are over 60 years of age. Movement of these huge numbers of returning pilgrims might pose a potential risk of transmitting MERS-CoV in Bangladesh and other countries across the world. Taking all these into account, the International Health Regulations (IHR) Emergency Committee advised strengthening MERS-CoV surveillance capacities to ensure timely reporting of any identified cases in countries with returning pilgrims. The Institute of Epidemiology, Disease Control & Research (IEDCR) under the Bangladesh Ministry of Health and Family Welfare (MoHFW) is mandated to lead outbreak investigations, surveillance and disease containment in the country, hence responsible for MERS-CoV screening in pilgrims/ travelers returning from the Middle East with respiratory illness (S1 Text).

A study conducted in the United Kingdom reported that respiratory infections among returning pilgrims were mainly caused by influenza and other respiratory viruses, such as Adeno virus, Respiratory syncytial virus, Human metapneumovirus and Para influenza viruses other than MERS-CoV. To determine if this is the case for Bangladesh, this study was conducted to detect the viral pathogens responsible for respiratory infections among returning Bangladeshi pilgrims and other travelers from the Middle East.

In June 2012, a patient with a severe respiratory infection was admitted to a hospital in Saudi Arabia. Known respiratory pathogens were ruled out as aetiological agents of the syndrome and the patient died of progressive respiratory and renal failure. Respiratory samples were further analysed, which resulted in the identification of a novel betacoronavirus: Middle East respiratory syndrome coronavirus (MERS-CoV) 1. As of 12 September 2013, a total of 114 laboratory-confirmed cases of MERS-CoV infection, including 54 deaths, have been reported (http://www.who.int/csr/don/2013_09_07/en/index.html). MERS-CoV is related to bat coronaviruses 2, although the animal reservoir has not been identified and the virus has been only isolated in humans. The severity of the infections and the ability of the virus to be transmitted person to person 3,4 raised international concerns about the possible spread and potential impact of the novel virus on human health. Asymptomatic or mild cases of MERS-CoV have been described 3,5, as well as cases of co-infection by MERS-CoV with other respiratory pathogens (http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_investigation_guideline_Jul13.pdf). Two recent studies assessed the transmission potential of the virus by mathematical modelling using currently existing data. Although the estimation of the inter-human transmissibility suggests that the virus is currently unlikely to cause a pandemic 6, it was also indicated that more than half of the symptomatic human cases would remain undetected 7.

Although most cases have been identified in Saudi Arabia, imported cases in European countries have also been reported. All imported cases had a travel history to the Middle East and therefore current recommendations for investigation of MERS-CoV cases include residence in or history of travel to Middle Eastern countries (http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_investigation_guideline_Jul13.pdf). However, the fact that the virus can be transmitted person to person and also cause mild disease could potentially result in unnoticed importation and circulation of the virus in other parts of the world apart from the identified geographical areas. Direct traffic from the Barcelona international airport includes 30 weekly flights to the Middle East, accounting for more than 600 000 passengers in 2012 (http://www.bcnair-route.com/index.php/en/intercontinental-flights). To explore whether MERS-CoV could have been introduced into our region and be circulating at a certain level, we screened respiratory samples from both mild and severe cases of acute respiratory infection, independently of the travel history of the patients.

The acute respiratory infections surveillance programme in Catalonia, PIDIRAC (daily information system for acute respiratory infections in Catalonia), performs surveillance during the influenza activity season from October to May (http://www.gencat.cat/temes/cat/salut.htm). This surveillance system is based on 38 sentinel primary-care centres in which physicians collect nasopharyngeal swabs from patients with acute respiratory infections as well as 11 sentinel hospitals that report cases of severe confirmed influenza virus infection admitted to their facility. Samples from the primary-care centres were screened at our laboratory for respiratory viruses using multiplex nested RT-PCR assays 8,9 that allow detection of influenza viruses A, B and C, respiratory syncytial virus, parainfluenza viruses 1–4, adenoviruses, human coronaviruses 229E and OC43, rhinoviruses and enteroviruses. This surveillance network provides timely information about the circulation of respiratory viruses in our region and carries out virological surveillance for the European Influenza Surveillance Network (EISN). For our study, we selected cases of mild respiratory infections attending the primary-care centres as well as patients with severe febrile respiratory syndromes admitted to our hospital who underwent bronchoalveolar lavage. A total of 563 samples from 563 corresponding patients were tested, from which 195 were collected between January 2012 and June 2012 and 368 between July 2012 and April 2013. We selected 304 nasopharyngeal swab samples from the surveillance network that were negative for respiratory viruses and 39 bronchoalveolar lavage samples that were negative for standard microbiological tests. Given that co-infections with other respiratory pathogens have been detected in patients with MERS-CoV infection, 200 nasopharyngeal swab samples in which a respiratory virus was identified and 20 bronchoalveolar lavage samples in which a respiratory pathogen was identified were also tested. The following viruses were detected among the positive nasopharyngeal samples: influenza viruses (51.8%), respiratory syncytial virus (17.4%), mixed viral infections (16.5%), adenoviruses (4.8%), coronaviruses (3.7%), parainfluenza viruses (3.4%) and enteroviruses (2%). Among the positive bronchoalveolar lavage samples for respiratory pathogens, 35% were positive for bacteria, 25% were positive for viruses, 20% were positive for fungi and 20% were mixed infections. RNA extraction of the respiratory samples was performed using an automated system (QiaSymphony; Qiagen, Hilden, Germany). A recently published real-time RT-PCR protocol for MERS-CoV 10 was implemented in our laboratory, and evaluated using an in vitro transcribed RNA kindly provided by C. Drosten through the European Virus Archive platform (http://www.european-virus-archive.com). The sensitivity of the assay in our hands was similar to that described by Corman et al. 10 (five RNA copies per reaction). In addition, we established a generic coronavirus RT-PCR as a confirmatory assay 11. This protocol further identifies the coronavirus species by sequencing of the PCR product. This assay was evaluated with samples containing 229E and OC43 human coronaviruses previously detected at our laboratory 12. All 563 respiratory samples tested were negative for the MERS-CoV using the real-time RT-PCR protocol.

The emergence of MERS-CoV represents a public health concern, although the virus does not seem to be as efficiently transmissible between people as the severe acute respiratory syndrome coronavirus. Many questions regarding the epidemiology of the virus remain unanswered, highlighting the importance of continued surveillance and research on MERS-CoV. To our knowledge, this is the first exploratory study of MERS-CoV in the general population using already established sentinel networks. Data obtained through active surveillance for MERS-CoV may be helpful for the global monitoring of the virus. Moreover, our results may serve as baseline reference data in case the virus starts to circulate in Catalonia, northeastern Spain.