Pertussis (whooping cough) is a highly contagious, respiratory disease caused by Bordetella pertussis (B. pertussis). The clinical symptoms of pertussis change with age, previous exposure to B. pertussis and immunization status. In newborns clinical manifestations may be severe. Most infants have a typical paroxysmal cough which can last more than two months.

Pertussis is a major cause of morbidity worldwide and of mortality in infants in developing countries. Pertussis continues as a public health concern threat given its re-emergence despite high vaccination coverage. Epidemic cycles reoccur every 2 to 5 years and 2015 has witnessed the worst outbreak in the past 70 years.

Although ample evidence confirms coinfections between B. pertussis and other pathogens, especially viruses, the role of coinfections remains debated [4–6]. Most mixed infections probably arise accidentally and whether they cause more severe disease than B. pertussis alone remains unclear [7–14]. Extending current knowledge on virus coinfections would make it easier to care for infants with pertussis.

We designed this study to compare clinical disease severity in infants with B. pertussis infection alone and those with B. pertussis and viral coinfections hospitalized in two Italian centers over two years. We also analyzed how respiratory infections and pertussis cases were distributed during the two years study. As primary outcome measures we assigned each infant a clinical severity score and assessed length of hospitalization. As an experimental approach to provide reliable data on lower respiratory virus infections we used an extended respiratory virus panel that can detect 14 respiratory viral targets with real-time reverse-transcriptase-polymerase chain reaction (RT-PCR) assay.

Acute respiratory infections (ARIs) remain one of the most common major public health threats, accounting for millions of episodes of severe acute lower respiratory infections that result in hospital admissions of otherwise healthy infants and young children worldwide. One-third of the annual deaths occurring in the world are thought to be due to infectious diseases, and respiratory tract infections are responsible for 4 million deaths worldwide each year. According to estimates made by the World Health Organization (WHO), pneumonia kills more children worldwide than any other disease, even more than acquired immune deficiency syndrome (AIDS), malaria and measles combined.

In healthy children, nasopharyngeal colonization with respiratory bacteria is a prerequisite for the development of respiratory or invasive (i.e., sepsis, meningitis) diseases. Asymptomatic transient nasopharyngeal colonization with bacteria, such as Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus, is common and decreases with age and the maturation of the immune system. Geographic region, ethnicity, season, day-care attendance, environmental factors and previous vaccinations are important determinants of bacterial colonization.

Respiratory viruses including influenza viruses, respiratory syncytial virus (RSV), human rhinoviruses (HRV), human metapneumovirus (HMPV), parainfluenza viruses, adenovirus (ADV) and human bocavirus (BoV) are responsible for approximately 35–87% of ARIs in children. Viral co-infections occur in 4–33% of children hospitalized with ARIs. Bacterial infections caused by S. pneumoniae and H. influenzae may commonly be observed in the later stages of respiratory diseases. The incidence of respiratory viral/bacterial co-infection in young children ranges from 1% to 44%, and studies on influenza pandemics over the last 100 years have strengthened the association of bacterial super-infections and influenza infections. In addition, pertussis and measles still represent serious medical issues with lower respiratory tract involvement in several countries.

There has recently been an increase in the number of available vaccines against respiratory pathogens recommended for children and adolescents by the health authorities, and many studies have been performed to evaluate their efficacy, safety and tolerability. The aim of this review is to report current issues about vaccines against some respiratory pathogens to highlight the available strategies to reduce the burden of paediatric respiratory disease.

Pertussis, also known as whooping cough, is an acute respiratory tract infection caused by Bordetella pertussis. Although the incidence rate of pertussis has significantly decreased following the wide use of pertussis vaccine, B. pertussis infection is continuously occurring as a small outbreak even in several developed countries with high vaccination coverage (1-4). There has been the resurgence of reported pertussis cases primarily in infants younger than 1 yr old, especially less than 3 months of age, in adolescents and young adults worldwide (5, 6). The possible causes of increased incidence in adolescents and adults include waning of vaccine immunity, adaptation of circulating B. pertussis strains, development of diagnostic methods, and active surveillance due to increased awareness of pertussis (7-9). Household contact with infected adolescents and adults also becomes the major source of pertussis infection in infants who are not fully immunized, and this problematic circulation may consequently threaten overall public health (10, 11).

The epidemiological characteristics of pertussis can vary depending on the definition of case, diagnostic method of confirmed case, national reporting system, network organization for epidemiological investigation, and the local vaccination schedule. National immunization program (NIP) of Korea consists of three primary series of diphtheria-tetanus-acellular pertussis vaccine (DTaP) at 2, 4, and 6 months, followed by a first booster at 15-18 months and a second booster between 4 and 6 yr of age. Recently, a tetanus toxoid, reduced diphtheria and acellular pertussis (Tdap) booster vaccine in adolescents aged 11-12 yr is added to Korean NIP in 2012. The vaccination rate of primary DTaP continues to be approximately 94% (12, 13), and there is a mandatory notification system in Korea. An annual average of about 11.5 cases of pertussis has been reported to the Korea Center for Disease Control and Prevention (KCDC), however, the reported cases of pertussis have increased since the 2000s. In addition, the KCDC reported that the incidence of pertussis was markedly increased (more than about 5.5 times) in 2009, compared to the previous (14). Also, the incidence of pertussis in 2011 was more than two times compared with the incidence in 2009 (15). We may predict the substantial outbreak in the future in our country.

Given the importance of accurately determining the epidemiological features of infant pertussis and the lack of reliable existing data in Korea, this study was conducted to describe the clinical characteristics of laboratory confirmed cases less than 1 yr of age and to evaluate the relative importance of family members on infants who are vulnerable to B. pertussis transmission.

Pertussis is a major public health problem, affecting adolescents and adults as well as children. Despite a widespread vaccination program, over the past fifteen years was seen a return of pertussis worldwide. Pertussis resurgence in Europe has been attributed to an incomplete immunization program or to genetic changes in Bordetella pertussis (B. pertussis). The currently used acellular pertussis vaccine contains the pertactin gene variant prn1 and the pertussis toxin-B S1 subunit. Molecular changes in these two genes over the past years suggest that the antigenic divergence may make pertussis vaccination less effective than before.

In some countries pertussis immunization is not mandatory and in others vaccination schedules suggest the first dose to be given at the age of 3 months. Hence, some infants remain unimmunized or incompletely immunized. In those who are incompletely immunized pertussis may develop in an atypical clinical form and be difficult to diagnose. Pertussis can be especially difficult to diagnose in children under 1 year of age during winter season, when other pathogens, such as respiratory syncytial virus (RSV), circulate. In these difficult cases, pertussis acute respiratory symptoms can overlap with those of bronchiolitis. A study conducted in a group of infants hospitalized for RSV bronchiolitis showed that almost 2% of the patients were co-infected with B. pertussis[7,8]. Since B. pertussis-RSV co-infection is infrequent in young infants, physicians should keep the possibility of co-infections in mind as to diagnose it early and prevent bronchiolitis from becoming more severe.

Although the standard diagnostic criterion for identifying B. pertussis is culture obtained from nasal swabs or nasopharyngeal aspirates, confirmatory information comes nowdays from molecular techniques such as real time-polymerase chain reaction (RT-PCR). Usually, in clinical practice the diagnosis is generally reached without microbiological confirmation. What we conspicuously lack is the clinician’s awareness of the clinical and laboratory data needed to reach a suspected B. pertussis diagnosis in order to start treatment early.

The main purposes in our retrospective, single-center study were to describe and compare clinical and laboratory features in infants with pertussis infection to infants hospitalized for RSV bronchiolitis, and to analyze the genetic characteristics of B. pertussis.

Acute bronchitis is an inflammation of the large airways that is characterized by cough and/or sputum that usually lasts one to three weeks. It is one of the most common illnesses among outpatients, and many patients receive antibiotic therapy [1–3].

Traditionally, viruses have been considered the main causative agent of acute bronchitis, possibly explaining the limited benefits of antibiotics [3–5]. However, data regarding the causative microorganisms are still limited. In previous studies, viruses were isolated in 8–23% of community-based cases, not frequently enough to conclude that viruses are the main causal agents for acute bronchitis. Macfarlane et al. identified viruses in only 19% of patients, while typical and atypical bacteria were identified in 25.9% and 23.7% of patients, respectively. In other studies, bacteria were detected in sputum samples in 45% of acute bronchitis patients [8, 9]. In addition, several authors suggested that some patients with acute bronchitis had mixed infections involving both viruses and bacteria. However, the exact prevalence and clinical characteristics of mixed infections have not been well studied. Moreover, it is not clear which subgroup of patients with acute bronchitis could benefit from antibiotic treatments. Recent big data from the UK show that antibiotics substantially reduce the risk of pneumonia after acute bronchitis, particularly in elderly people in whom the risk is highest.

Therefore, in the present study, we aimed to investigate the frequencies and characteristics of viral, bacterial, and mixed infections in acute bronchitis in the community. We also hypothesized that the frequencies of these etiologies would vary with underlying lung co-morbidities and age.

B. pertussis DNA was extracted with QIAamp DNA minikit (QiaGEM, Hilden, Germania) and amplified with the “Bordetella Real-Time PCR” kit (Diagenode Diagnostics, Liège, Belgio). The assay gave binary results. For RT-PCR the SYBR Green Detection assay we used the LightCycler 2.0 system (Roche Diagnostic). Data were analyzed with LightCycler software (version 4.0, Roche Diagnostic). Only the positive samples for B. pertussis were cultured on charcoal agar plates (Oxoid England) containing defibrinated sheep blood at 10% and incubated at 35 °C up to 7 days and inspected daily, as previously described.

The study was conducted in the PICU of Children’s Hospital Béchir Hamza of Tunis. The PICU is in a university-affiliated children’s hospital and provides intensive care services to a national pediatric population of 850 000 children less than 15 years old. The hospital has 360 beds, and the PICU has 16 beds (500 admissions/year).

Adult patients with acute bronchitis were prospectively recruited at 31 Korean hospital outpatient departments and primary clinics between July 2011 and June 2012 (6 university-affiliated teaching hospitals, 5 non-teaching community hospitals, and 20 primary clinics). Sputum samples for Gram stains, conventional cultures, and polymerase chain reaction (PCR) were collected from each patient before any medications (including antibiotics) were prescribed. Medications were chosen at the physicians’ discretion. The study protocol was approved by the Institutional Review Board of Hallym University Sacred Heart Hospital (the principal institute, 2011-I049) and each participating hospital. All participants provided informed written consent.

Patients were eligible if they were ≥18 years old and visited the outpatient clinic because of cough (duration < 1 month) with sputum production. Acute bronchitis is a clinical diagnosis, and therefore, a wrong diagnosis is possible. Coughing symptom may have almost all respiratory illnesses as a differential diagnosis. However, symptoms such as sputum production, after carefully discriminating from postnasal drip, could also lead to a diagnosis of lower respiratory inflammation. Patients with typical upper respiratory infection (URI) and symptoms of influenza or influenza-like illness (ILI) during the epidemic period were excluded by participating physicians. We tried to rule out URI by conducting detailed medical interviews, throat examination, and by auscultation. Typically, URI was defined as an infection affecting patients presenting with key symptoms such as sore throat, and nasal symptoms (nasal obstruction, runny nose) with cough. ILI was defined as an abrupt onset of fever with non-productive cough or sore throat. The period of the influenza epidemic was determined by means of a national respiratory virus surveillance system which was broadcast weekly. In some cases, chest radiographs were done at the investigating physician’s discretion, in order to rule out pneumonia. Other exclusion criteria were: 1) history of antibiotic treatment < 7 days before the visit, 2) exacerbation of chronic lung disease within 6 months, 3) active lesion on the chest or paranasal sinus radiographs (when available), 4) immunocompromised status, and 5) confirmed alternative cause for the cough (e.g., drugs [newly started on angiotensin-converting enzyme inhibitors], pneumonia, allergic rhinitis, sinusitis, or gastro-esophageal reflux). Stable chronic lung disease patients were not excluded.

Chronic cough is defined as daily cough lasting 4 weeks or longer.1 In children under 15 years of age, the causes of chronic cough include congenital defects, asthma, foreign body aspiration, infections, allergic diseases, gastro-esophageal reflux, tumors, and other rare causes.2 Among these, the most common etiologies include asthma, upper airway cough syndrome, protracted bacterial bronchitis, and nonspecific cough.3,4 The diagnosis of asthma or cough variant asthma can be made on the basis of NAEPP EPR-3 criteria and response to a bronchodilator.5 Upper airway cough syndrome is a common entity,3,4 most likely triggered by postnasal drip due to either allergic and nonallergic rhinitis or bacterial and nonbacterial sinusitis. Protracted bacterial bronchitis is now recognized as one of the most common causes of chronic wet cough, especially in young children under 5 years of age.6,7

Once asthma and other conditions listed above are ruled out, upper airway cough syndrome or protracted bacterial bronchitis need to be considered as the most likely diagnoses. In approaching these clinical entities, our goal was to promptly detect the patients with bacterial sinusitis and the involved pathogens and to treat them with appropriate antibiotics. To this end, we decided to employ a novel method described by Huang and Small,8 who reported that nasal secretion dipstick assay was highly correlated with sinus imaging studies and could be utilized for the diagnosis of sinusitis. We employed this simple approach for obvious reasons: speed, cost-effectiveness, and avoidance of radiation risk associated with sinus imaging studies. We also sought an equally simple and rapid method for identification of the pathogens for the suspected sinusitis. Positive findings in imaging studies of sinuses such as opacities and thickened lining indicate the presence of inflammation, possibly infection, but may have poor correlation with clinical disease9,10 and do not reveal any information on etiologies. Although sinus puncture would yield such information,11 this cannot be performed in most nonsurgical office settings. Studies have documented that nasal culture is a poor reflection of the microbes in the sinus cavity.12 Therefore, we analyzed the microbes from the osteomeatal area utilizing a quantitative PCR method in an attempt to detect the pathogens that may cohabit both sinus and nasal cavities.

Pertussis is an acute respiratory illness caused by Bordetella pertussis (B. pertussis). Critical pertussis (CP) is defined as pertussis disease that results in pediatric intensive care unit (PICU) admission or death. It is characterized by severe respiratory failure, important leukocytosis, pulmonary hypertension, septic shock and encephalopathy. Despite intensive care management, it causes substantial morbidity and mortality for children especially among young infants. Resurgence of pertussis in the last 20 years is evident from the Centers for Disease Control (CDC).1 Several reasons for this resurgence have been proposed, including genetic changes in Bordetella pertussis, lessened potency of pertussis vaccines, waning of vaccine-induced immunity, greater awareness of pertussis, and the general availability of better laboratory tests.2 A new resurgence was seen in 2013 in Tunisia even in the presence on a high (98%) coverage of childhood vaccination.3 The purpose of this study was to describe the institutional experience in the management of infants with CP admitted in year 2013 at Children’s hospital Bechir Hamza of Tunis, reporting the relationship between method of presentation, therapies and outcome in order to identify factors associated with death.

We studied 7 patients with rhinitis and positive skin tests for respiratory allergens but without cough and lower respiratory symptoms.

Despite a widespread vaccination program, pertussis continues to be a common worldwide infection in pediatric and adult populations. In the past decade, there has been a resurgence of this disease in United States and European countries, peaking every 2 to 5 years [1–7]. The last peak reported in Europe was in 2012.

In contrast to what is reported in other countries, in Italy after the introduction of acellular vaccine in 1995 incidence has continued to decrease and pertussis has not reemerged yet [9, 10]. Therefore epidemic cycles have been clearly less identifiable due to the low incidence. In our country, vaccination schedule provides a pertussis vaccine dose at 3, 5 and 12 months and a booster is recommended in the preschool period and in adolescents. Vaccination coverage during the analyzed period was around 95 %.

Since other countries with high immunization coverage over a long period of time experienced a resurgence of pertussis [3, 5], we hypothesized that the epidemiology of this disease in Italy may be affected by the lack of recognition by clinicians with the consequence of limiting the use of laboratory confirmation. In clinical practice the diagnosis of pertussis is generally reached without microbiological confirmation leading to a possible lack of clinical awareness to start early treatment and prevent complications.

Infants are known to acquire pertussis from adolescents’ and adults’ contacts that return susceptible to the disease because of waning immunity as well as from unvaccinated children [12–14]. Clinical manifestations may be different depending on age. Severe symptoms are common in young unvaccinated infants and pertussis continues to be a major cause of vaccine-preventable death in this age group. However, cases with atypical clinical presentations do occur and may be often unrecognized, especially during the winter season, when other respiratory viruses circulate and the minimum incidence of pertussis is usually observed. The annual seasonality in the Italian pertussis incidence peaked between March and August while the minimum incidence has been observed between September and February.

We therefore systematically studied a series of infants ≤3 months of age hospitalized with respiratory symptoms to detect how frequently physicians suspected pertussis on a clinical basis and the actual frequency of laboratory confirmed cases. We compared patients with pertussis infections and patients with other respiratory infections to identify clinical and laboratory predictors of pertussis.

Influenza A virus (IAV) is a major animal and public health concern given the zoonotic nature of IAV (1–3). As a natural host to IAV, research on IAV in swine has relevance to both human and animal medicine. IAV is a segmented, negative-sense, single-stranded RNA virus. The surface glycoproteins, hemaggluttinin (HA) and neuraminidase (NA), are used to type IAV, and currently H1N1, H1N2, and H3N2 viruses circulate in pigs in the United States (4, 5). There are a large number of IAV H1 and H3 genetic and antigenic variants co-circulating, and continued antigenic drift and shift of circulating viruses has made control of IAV in swine very difficult (5). Live-attenuated influenza virus (LAIV) vaccination in swine has been shown to provide cross-protection against heterologous IAV of the same subtype, and partial protection against different subtypes [reviewed in Sandbulte et al. (6)]. LAIV is licensed for use in humans and was recently approved for use in swine, with numerous experimental studies documenting improved efficacy of LAIV over inactivated vaccines (7, 8). Several LAIV vaccines for use in swine have been developed; each with a different attenuation mechanism (9–11). Similar to humans, intranasal LAIV vaccination in pigs induces the production of IAV-specific mucosal IgA, but little peripheral IAV-specific IgG (8). The induction of immunity in the respiratory tract has been shown to be the mechanism by which LAIV vaccines provide significant cross-protection against heterologous strains of IAV, limiting viral replication throughout the respiratory tract [reviewed in Rose et al. (12)].

Bordetella bronchiseptica can colonize the respiratory tract of a large number of mammals, including mice, rabbits, dogs and pigs, among others. Respiratory disease associated with B. bronchiseptica covers a wide spectrum, including kennel cough in dogs and atrophic rhinitis in pigs (13, 14). In humans, B. pertussis infection can lead to whooping cough, though colonization without clinical presentation has been documented (15, 16). Similarly, B. bronchiseptica exposure to pigs can result in chronic, asymptomatic colonization of the respiratory tract and it is believed to be ubiquitous in swine production systems. Co-infection with IAV or coronavirus and B. bronchiseptica in pigs causes exacerbated pulmonary disease, indicating the negative impact of B. bronchiseptica colonization with viral infection (17, 18). Bordetella species encode for a number of virulence factors, including tracheal cytotoxin, dermonecrotic toxin, lipopolysaccharide, and a type III secretion system (19). While the gene locus controlling expression of many virulence factors, including the type III secretion system, has been highly investigated, factors that alter expression of virulence genes in vivo are not completely understood (20, 21).

In the past decade, the complex interaction between mucosal surfaces and colonizing microbiota has been recognized as important in modulating both health and disease states [reviewed in Esposito and principi (22)]. The commensal microbiota of the upper respiratory tract includes bacterial species in which colonization alone does not lead to clinical disease, but upon a stressful event (i.e., viral infection, immunosuppression) these bacteria play a major role in disease pathogenesis, often referred to as pathobionts. Administration of LAIV vaccine induces changes in the nasal microbiota and gene expression in nasal epithelium. In addition, LAIV administration alters colonization dynamics of important bacterial pathogens (23). Given the ubiquitous nature of B. bronchiseptica in swine and the documented increase in disease following B. bronchiseptica with IAV co-infection, we performed a study to determine if B. bronchiseptica colonization prior to LAIV vaccination altered LAIV immunogenicity and efficacy against heterologous IAV challenge, or the dynamics of B. bronchiseptica colonization.

During the study period, we enrolled 215 patients. The admission diagnosis of those patients is reported in Table 1.

Out of 215 patients tested, 53 had a positive RT-PCR for BP (24.7 %). Of the 162 patients resulted negative for BP, 119 were positive for RV infections (55.3 %): RSV was diagnosed in 48 (40.3 %), Rhinovirus in 37 (31.1 %), Parainfluenzae Virus in 9 (7.6 %), Adenovirus in 4 (3.4 %), Metapneumovirus in 4 (3.4 %), Influenzae Virus in 3 (2.5 %), Coronavirus in 3 (2.5 %), Rhinovirus + Adenovirus in 4 (3.4 %), Rhinovirus + Coronavirus in 2 (1.7 %), RSV + Adenovirus in 2 (1.7 %), Parainfluenzae virus + Metapmeumovirus in 2 (1.7 %), RSV + Coronavirus in 1 (0.8 %).

No etiological agent was identified on nasopharyngeal aspirate of 43 patients (20 %). Those patients were discharged with the following diagnoses: apnea (ICD-9 code 78609) (21), bronchiolitis (13), laryngitis/laryngomalacia (3), unexplained fever in infants (2), sepsis (1), pneumonia (1), intraventricular septal defect (1), HHV6 encephalitis (1).

At admission, pertussis was clinical suspected in 22 patients only on the basis of the WHO definition. Sixteen of them had a positive RT-PCR for BP, while the 6 patients resulted negative to BP were discharged with diagnosis of bronchiolitis in 5 cases (2 RSV, 2 Rhinovirus and 1 Parainfluenzae Virus) and apnea in 1 case (negative nasopharyngeal aspirate). On the other hand, among the remaining 193 patients who had a different diagnosis at admission, 37 were RT-PCR positive for BP. Thus the sensitivity of clinical diagnosis at admission was 30.2 % (19.52–43.54) and the specificity 96.3 % (92.16–98.29).

The clinical and laboratory characteristics on admission were compared between BP+ patients, RV+ patients and BP-RV- patients (Table 2). Cough, paroxysmal cough, whoop, apnea, fever, rhinorrhea, white blood count, lymphocytes count, length of symptoms before admission and length of hospital stay were statistically different among the three groups.

When we applied the logistic regression model to explore predictive clinical manifestations and/or laboratory test for pertussis, data showed that paroxysmal cough, absence of fever, absolute lymphocyte count >10.000 n/mm3 and duration of symptoms before admission ≥5 days were significantly associated with pertussis compared with other diagnoses (Table 3).

Notably, when we analyzed the length of symptoms before admission of patients with BP+ we found that 20 patients (37.7 %) reported symptoms for less than 7 days; 20 patients (37.7 %) reported symptoms for 7- < 14 days and 13 of them (24.5 %) reported symptoms for more than 14 days.

Therefore, our data showed that apnea is not predictive for pertussis, but it’s a frequent clinical manifestation (30/53); among BP+ patients, 22 (41.5 %) reported apnea associated with cough and cyanosis, while 8 of them (15.1 %) reported apnea alone not associated with other symptoms.

Complications (oxygen requirement and pneumonia) were not statistically different in the three groups. No deaths were reported (Table 2).

Regarding ongoing antibiotic therapy, 34 of our patients (9 BP+, 21 VR+, 4 BP-/VR-) had already started antibiotics before admission; particularly, 19 patients (7 BP+, 10 VR+, 2 BP-/VR-) had already started macrolide therapy when specimens were collected.

When we analyzed the seasonal trend of our BP+ patients, we found that the maximum incidence was between June and September, but we had cases even in winter with a peak in February (Fig. 1).

With regard to coinfection, of the 53 pertussis cases, 18 (34 %) had a positive RV result in addition to BP: 8 patients had PCR positive for Rhinovirus, 4 for Coronavirus, 1 for RSV, 1 for Metapneumovirus, 1 for Parainfluenza Virus, 1 for Influenza + Coronavirus, 2 for Rhinovirus + Parainfluenza. We didn’t find any significant differences between patients with pertussis as monoinfection and patients with pertussis plus RV infection.

Pertussis is a highly contagious respiratory tract infection, caused mainly by Bordetella pertussis and less frequently by Bordetella parapertussis. In the pre-vaccination era, infants and children contracted pertussis in their first years of life, with a clinical course characterized by uncontrollable coughing attacks, often accompanied by paroxysms, post-tussive vomiting, and inspiratory whooping. Consistently high vaccination coverage has substantially decreased pertussis in the population [2, 3], but newborns too young to be vaccinated remain at high risk for severe complications including apnea, cyanosis, pneumonia, encephalopathy or even death. This risk is increasing due to the worldwide pertussis reemergence in the 1990s, even in areas of high vaccination coverage in all age groups, with transmission of disease from household members to newborns. Today, high pertussis incidences in infants are observed, with incidence peaking every two to three years [3, 5, 6]. Worldwide in 2014, an estimated 24 million cases and 160,000 deaths from pertussis occurred in children younger than 5 years, with the African region contributing the greatest share. In the Netherlands, each year approximately 150–180 children <2y are hospitalized and one infant, in general too young to be vaccinated, dies due to pertussis. For this reason, many countries are discussing prenatal pertussis vaccination of mothers to protect newborns, and a growing number of countries now recommend it. This measure is effective in preventing pertussis in the first months of life and has decreased the pertussis disease burden in young infants [10, 11]. In the Netherlands, the Health Council advised that 3rd trimester maternal pertussis vaccination be offered. This is overall very effective in prevention of pertussis in early infancy, but preterms may benefit less due to a smaller time-window for mother-to-child transfer of antibodies before delivery [12, 13]. However, vaccine effectiveness (VE) is reportedly lower after 2nd trimester pertussis vaccination. Given the introduction of a maternal vaccination strategy against pertussis in The Netherlands, we sought to gain more insight into the current pertussis burden among hospitalized infants, with special attention to preterms.

Respiratory syncytial virus (RSV) is the major infectious cause of lower respiratory tract illness in infants and young children around the world.1, 2 It has also been recognized as an important etiologic agent of pneumonia and other respiratory tract infections in adults and elderly patients.3, 4 The clinical presentation of this infection varies widely, from mild upper respiratory tract disease to bronchiolitis and pneumonia.5 This virus is responsible for the majority of bronchiolitis cases and causes approximately 50% of pneumonia cases during the first years of life.6 In children, host factors such as young age, prematurity, and chronic cardiopulmonary diseases have been associated with severe disease. In addition, other factors such as lower socioeconomic status, exposure to cigarette smoke, air pollution, crowded households, and the lack of breastfeeding have also been associated with severe disease.7 Viral factors associated with virulence leading to severe disease are not sufficiently understood.8

Human RSV is a member of the Paramyxoviridae family. Outbreaks of RSV infections occur between fall and spring in temperate climates and tend to last up to 5 months.9, 10 RSV isolates can be divided into two groups: group A and group B based on antigenic and genetic characteristics.8 These two groups cocirculate in the human population, with group A being more prevalent. Several studies have compared the severity of disease between infants infected with RSV group A and group B with mixed results. Most studies have not found significant clinical differences between both subtypes.8 However, a possible effect of different viral strains on disease severity remains an open question.

Despite the recognized importance of RSV as a cause of respiratory illness, the information regarding the epidemiology of this virus in Latin America, particularly among adults, is limited.11 In the present study, 570 cases of RSV infection identified during four epidemic years in Mexico were evaluated to clarify the epidemiology of this infection and to assess the possible variations in demographic and clinical characteristics according to viral groups.

Coronaviruses (CoVs), a genus of the Coronaviridae family, are positive-stranded RNA viruses. The first human coronavirus (HCoV) appeared in reports in the mid-1960s and was isolated from persons with common cold. Two species were first detected: HCoV-229E and subsequently HCoV-OC43 [1, 2]. Since then, more species were described [3–5].

The HCoV-229E strain was associated with common cold symptoms. Younger children and the elderly were considered more vulnerable to lower respiratory tract infections. Severe lower respiratory tract infection so far has only been described in immunocompromised patients [7, 8]. To our knowledge, there is no report describing life-threatening conditions in immunocompetent adults attributed to HCoV-229E. We report a case of acute respiratory distress syndrome developed in a healthy adult with no comorbidities and HCoV-229E strain identified as the only causative agent.

Nasopharyngeal cultures, polymerase chain reaction (PCR) testing and serologic studies are available to confirm an infection with Bordetella pertussis, the causative organism.11 However, these tests offer varying levels of sensitivity and may not be obtainable in a timely fashion to confirm cases in the acute setting. Furthermore, other laboratory studies, such as a complete blood count (CBC), may be helpful in distinguishing causes for cough, but only in certain age groups (see “Differential Diagnosis” section). Imaging studies also provide limited information, as patients often do not demonstrate significant findings on chest radiograph. However, chest imaging may be helpful in assessing for superinfection.

Acute respiratory infections (ARIs) are a leading cause of morbidity, hospitalization, and mortality among children [1–3]. According to World Health Organization (WHO), acute respiratory infections are responsible for 1.9 million annual deaths in children, mainly affecting patients under 5 years old, with a higher incidence in those from low-income countries [1, 4].

ARIs are mainly caused by a wide range of viruses and bacteria [5, 6]. Viruses are isolated in up to 80% of cases, the most common pathogens are the respiratory syncytial virus (RSV) A and B, influenza (Flu) A, B and C, parainfluenza (PIV) types 1, 2, 3 and 4, coronavirus and rhinovirus [7, 8]. Classically, S. pneumoniae and H. influenzae type b are the most commonly isolated bacteria in both throat and nasopharyngeal specimens from patients with ARIs [9, 10]. However, in resource-limited countries, atypical bacteria such as Mycoplasma pneumoniae, Chlamydia pneumoniae, and Bordetella pertussis can play an important role in ARIs and can be detected in more than 40% of patients [2, 11–14].

Although numerous pathogens are associated with ARIs, their clinical manifestations are very similar, regardless of the causative agent. Thus, laboratory identification of the etiological agent is key in order to give a proper treatment and avoid the overuse of antibiotics. Moreover, ARIs due to atypical bacterial infections have become a global concern especially after their reemergence in low-income countries [11, 16].

Simultaneous infections with virus and bacteria species have become an obstacle for clinicians, their prevalence has significantly increased, with studies discovering co-infections in more than 45% of cases [11, 17–19]. Additionally, these coinfections have been associated with longer hospitalization periods, worse clinical outcomes and increased mortality, again highlighting the importance of molecular etiological confirmation [17, 19, 20].

Bordetella pertussis represents a persistent cause of morbidity and mortality in children. Accounting for an estimated 16 million cases and 195,000 deaths worldwide. In a previous study we conducted on children under 1-year-old with a probable diagnosis of Pertussis from 5 Peruvian hospitals, we reported a prevalence of 39.54% pertussis cases. With more than 60% of cases without an identified pathogen, hence a more comprehensive etiological analysis was required.

The main objective of this study was to detect the presence of 8 respiratory viruses (Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza-1, Parainfluenza-2 and Parainfluenza-3) and atypical bacteria (Mycoplasma pneumoniae, Chlamydia pneumonia), via Polymerase Chain Reaction in samples from Peruvian children under 5 years-old previously analyzed for B. Pertussis.

Severe and sometimes fatal pertussis-related complications can occur in certain groups. These include infants <12 months of age, particularly those Often, patients become secondarily infected with another bacterial or viral infection. Neonates are especially at risk for apnea and hemodynamic instability (i.e., bradycardia, hypotension). Although rare, seizures and encephalopathy can also occur.12,17,19

Acute respiratory tract infections (RTIs) are the leading causes of outpatient visits and hospitalizations in all age groups, especially during winter and spring. For children under 5 years of age, RTIs are the second leading cause of death. Most acute RTIs in children are caused by respiratory viruses, such as respiratory syncytial virus (RSV), adenovirus (ADV), rhinovirus (RV) and influenza viruses. In addition to viruses, atypical pathogens are major causes of pediatric RTIs. One of the most common atypical pathogens is Mycoplasma pneumoniae (M. pneumoniae), accounting for 10–40% of hospitalized children with community-acquired pneumonia [2, 3]. In addition to M. pneumoniae, the incidence of pertussis in China has significantly increased since 2010. Nevertheless, multiple epidemiological studies have suggested that the incidence of pertussis in China has been significantly underestimated [4, 5]. The early diagnosis of the pathogen is beneficial for the precise selection of medication, which can largely avoid the overuse or even abuse of the antibiotics and improve the clinical care of patients. More importantly, the early diagnosis of contagious pathogens, such as Bordetella pertussis (B. pertussis) and influenza viruses, can enable early isolation of patients, thus reducing the spread of pathogens.

At present, the routine detection methods for respiratory pathogens in China are mostly based on immunological methods, which include the detection of M. pneumoniae and several major viruses, such as RSV, ADV, RV, parainfluenza virus (Para), influenza A virus (FluA) and influenza B virus (FluB). Other respiratory viruses and atypical bacteria, such as Chlamydophila pneumoniae (C. pneumoniae) and B. pertussis, are typically not routinely detected. Given their poor sensitivity and long turn-around time (TAT), immunological methods usually lead to broad-spectrum therapy and have been gradually replaced by molecular-based methods, such as conventional and real-time polymerase chain reaction (PCR), in developed countries [6, 7]. However, most of these molecular tests are technically challenging and require independent spaces, such as pre-PCR and post-PCR rooms, to eliminate the potential risk of cross-contamination, and such requirement limits their applications in China. Therefore, faster, more sensitive and easy-to-use assays for multiplex respiratory pathogen detection are urgently needed.

FilmArray (BioFire Diagnostics, Utah, USA, owned by bioMérieux) is a small, desktop, fully automated multiplex PCR device. The molecular system includes automated nucleic acid extraction, an initial reverse transcription step and multiplex nested PCR, followed by a melting curve analysis. The FilmArray Respiratory Panel (FilmArray RP) is both FDA-approved and CE IVD-marked. The current version of FilmArray RP (v1.7) is able to detect 16 viral and 3 atypical respiratory organisms. The test is performed in a closed system that requires 5 min of hands-on time and 65 min of instrumentation time. Several comparison studies between FilmArray and other tests for respiratory organisms showed comparable results [9–11].

The aim of this study was to evaluate the application of FilmArray RP for the detection of respiratory organisms, and to provide information about the seasonality and prevalence of these organisms in pediatric patients with RTIs in a large children’s hospital in China.

A prospective, multicenter, observational study was conducted between January 2009 and September 2011. Three hospitals participated in the study: Seoul St. Mary's hospital; Suwon St. Vincent's hospital; and Incheon St. Mary's hospital. We evaluated all infants clinically suspected of pertussis infection because of a cough lasting at least 2 weeks with at least one of the following symptoms: paroxysmal coughing; inspiratory whooping; post-tussive vomiting or apnea without other known cause. We obtained information on current respiratory manifestations, radiologic findings and immunization status for each infant. Diagnostic approaches for pertussis were conducted for clinical cases. Nasopharyngeal aspirates (NPA), or swab samples if aspirates were not possible, and blood samples were collected within 2 days at admission. Laboratory tests were performed at the Vaccine Bio Institute (VBI) of The Catholic University of Korea. It is determined as the criteria for laboratory-confirmed pertussis case when the subjects is applicable to one of the following criteria: 1) positive result of B. pertussis on culture of nasopharyngeal aspirates (NPA) or swab; this sample was collected and cultured on Regan-Lowe culture medium at 37℃ for more than 1 week; 2) positive result of B. pertussis in PCR or real-time PCR (RT-PCR) of NPA or swab; PCR was done by the method Glare et al. reported (16), and RT-PCR was done by modified method of Reischl U. Colleagues manual (17); 3) positive serology which defined as pertussis toxin (PT) antibody in a single serum sample that was higher than cut-off value (24 EU/mL) of enzyme-linked immunosorbent assay (ELISA) kit (IBL, Hamburg, Germany) or a 4-fold increased change in anti-PT antibody between acute-phase and convalescent-phase serum.

After the laboratory confirmation on pertussis, the parent or legal guardian for registration of index case was contacted as soon as possible, and all infants who are eligible for inclusion criteria were registered to the study immediately upon receipt of the consent form. To all family members if cough started at least 7 days before the onset of symptoms in the index case, it was requested to participate in the study as household contacts. And they were registered for the study immediately upon receipt of the consent form, and interviewed using the standard questionnaire to collect demographic and clinical data. Also, for all long-term household contacts, it was asked to visit for the study to collect respiratory samples (for culture, PCR, RT-PCR identification of B. pertussis) and/or serum samples (for ELISA).

The community-acquired pneumonia (CAP) is the leading cause of morbidity and mortality worldwide. According to WHO estimates, 450 million cases of pneumonia are recorded each year, with about 4 million deaths from this illness [1, 2], with the highest incidence cases in children younger than 5 years old. The mortality rate has declined greatly due to early and accurate detection of the etiological agents, together with the timely initiation of appropriate treatment.

Nowadays, the golden standard for CAP diagnosis is still based on the chest radiography; however, a broad range of chest radiographic changes could be induced by various agents, a single and dual bacterium/virus, or mixed pathogens coinfections. These alterations of chest radiography are only helpful in specific cases to confirm a microbial cause of pneumonia. Current diagnostic methods identify a pathogen in only 30%–40% of CAP patients [5, 6], and the frequent lack of a microbiological diagnosis in CAP impairs pathogen-directed antimicrobial therapy. Polymerase chain reaction (PCR) technique has been shown to be reliable for diagnosing the pathogens, especially for those that are difficult to culture. In addition, PCR method has high sensitivity and specificity in detecting multiple microorganisms and yields results faster than culture and serological methods. Most importantly, the results of PCR are not affected by prior use of antibiotics. The findings indicate that the incidence of viral pneumonia in the past has been underestimated. With multiple PCR assay, detection of multiple RNA or DNA targets in a single tube has the potential for rapid identification of complicated respiratory viral pathogens [9, 10]. The conventional techniques of microbial detection have high accuracy, but they are time- and labor-intensive, with limited range of detection and can be subjective, relying very much on technical expertise for the interpretation of cytopathic effect (CPE) in cell culture. Direct fluorescent antibody assay (DFA) and immunochromatographic antigen testing, although rapid, have poor sensitivity for the detection of most viruses. The serological methods also have low sensitivity. Therefore, an assay that is capable of rapid detection and accurate identification of multiple pathogens is desirable.

FilmArray Respiratory Panel (RP) is a multiplexed nucleic acid test for the simultaneous qualitative detection and identification of multiple respiratory virus, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae. Particularly, the whole process only takes about an hour; compared to multiple PCR, the FilmArray RP has provided fast results. As the FilmArray RP is emerging method, which is rarely employed in clinical specimen's detection, its clinical significance in diagnosing CAP is sparse. Therefore, in this study, we evaluated the capability of FilmArray RP for simultaneous identification of multiple pathogens; its microbial yield and clinical significance in CAP diagnosis were also evaluated.

The significance of finding pathogenic bacteria in the upper respiratory tract is unknown, as it is generally thought that many people are asymptomatic carriers of colonising bacteria [1–3]. To date, there has been very little research looking at the role of upper respiratory tract bacteria as a cause of mild respiratory illness. Healthcare workers (HCWs) are at high risk of nosocomial infection with respiratory, blood borne and other infections [4–6]. Respiratory infections are the leading cause of acute infections in humans. Whilst the burden of disease is well recognised in the elderly and immunocompromised, limited information exists on the impact of respiratory infections on HCWs [6–8]. Nosocomial infections among HCWs result in increased rates of illness, absenteeism and even death amongst HCWs [5, 9] and also comes at a large financial cost to the health care system. HCWs may also be a source of infection in nosocomial outbreaks. Transmission of influenza from patient-to-HCW and HCW-to-patient have been identified in many cases highlighting the importance HCWs play in hospital transmission.

Varying degrees of nasopharyngeal (NP) bacterial colonisation are reported in healthy individuals, depending on the age of participants, site of specimen collection and vaccination status [13, 14]. Certain factors increase the risk of nasopharyngeal carriage such as age, geographical area, vaccination status, immunity and socio-economic status [1, 13–15]. There are limited data from HCWs, and most studies are conducted in children, who generally have higher rates of NP colonisation compared to adults [3, 16]. Commonly isolated organisms are Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus. Testing of healthy children showed 50% NP carriage rate for S. aureus, 55–69% for Streptococcus pneumoniae [13, 14, 18], 67–74%for Moraxella. catarrhalis [13, 14] and 57–83% for Haemophilus influenzae [13, 14, 18].

In a study of both inpatients and outpatient children the overall swab positivity rates were 31.5% (63/200), with Streptococcus pneumoniae, Haemophilus influenzae and Group A Streptococcus accounting for 22%, 5% and 4.5% respectively. In a study in Italy, the rate of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis found 3.5%, 11.9% and 4.1% carriage respectively (overall 17.9%). In Australian Aboriginal adults, rates of bacterial colonisation were 26%, 23% and 17% for Streptococcus pneumoniae, Haemophilus influenza and Moraxella catarrhalis respectively. Lower carriage rates of Haemophilus influenzae (0%) and Streptococcus pneumoniae (10.8%) were observed in a study in China. An Australian study also reported very low rate (1/315) of Streptococcus pneumoniae in hospitalised elderly patients.

The finding of a virus in the upper respiratory tract is generally thought to be clinically significant. However the significance of asymptomatic viral infection has not been explored in existing literature, and viruses detected in the upper respiratory tract are generally assumed to be pathogenic. Some studies have found that up to 1 in 3 subjects with influenza infection may be asymptomatic, whilst asymptomatic infection with parainfluenza virus has also been observed. One study also demonstrated shedding of parainfluenza virus from healthy subjects over an extended period of time. Whilst little research exists on asymptomatic viral infections, these cases suggest that viruses can be transmitted by asymptomatic subjects unknowingly. A better understanding of the role of bacterial and viral infections in HCWs and their association with the respiratory symptoms is warranted. The aim of this study was to examine the presence and significance of bacteria and viruses in the upper respiratory tract of HCWs.

A 45-year-old female patient presented to the emergency department with dry cough, headache, and fever up to 39.5°C lasting a few hours. Her past medical history was unremarkable, and she did not take any medication regularly. She has never smoked, worked as a teacher at a local high school, and has not recently travelled.

Clinical examination revealed rales at her left lower lung fields. Chest X-ray showed diffuse opacities and consolidation at this field. The arterial blood gases (ABGs) were normal, and intravenous ceftriaxone and azithromycin were empirically administered for lower respiratory tract infection (LRTI). S. pneumoniae and L. pneumophila antigen in the patient's urine specimen was negative, and blood cultures were sterile.

Over the next two days, the patient's clinical condition rapidly deteriorated, with development of tachypnea (34 respirations/minute), dyspnea, and hypoxemia. ABGs changed to PaO2 of 55.3 mmHg, PCO2 of 31.4 mmHg, and pH of 7.487. Lung auscultation revealed diffuse rhonchi symmetrically all over her chest, bronchial breathing at her right and left lower lobes, and diminished vesicular sounds. Chest CT scan displayed bibasilar pleural effusions and diffuse consolidations plus ground glass opacities involving all lung fields (Figure 1). Oxygen was supplied at 5 L/min, and antimicrobial therapy was changed to levofloxacin 500 mg/day. Systemic corticosteroids and bronchodilators were added about 40 hours after her hospitalization. Samples of the pleural fluid showed exudate with 260 cells/mm3, negative Gram stain, and sterile cultures.

Nasal secretions were collected, and multiplex PCR technology was applied targeting multiple pathogens (RespiFinder® 22, PathoFinder), including coronavirus 229E; coronavirus NL63, HKU1, and OC43; influenza A, B, and H1N1; parainfluenza 1, 2, 3, and 4; Mycoplasma pneumoniae; Legionella pneumophila; Bordetella pertussis; bocavirus; rhinovirus/Enterovirus; adenovirus; RSV A and B; and Chlamydophila pneumoniae. The result was positive for HCoV-229E, while negative for the other tested pathogens; PCR for SARS-CoV and MERS-CoV was also negative.

Within the next few hours, the patient's clinical condition further worsened and she required increased oxygen supply. New ABGs showed PaO2 = 76 mmHg, PCO2 = 33 mmHg, and pH = 7.45 at FiO2 = 0.50 with PaO2/FiO2 = 152, indicating ARDS. The patient was in severe respiratory distress and remained febrile and tachypneic, and a new chest X-ray showed multiple consolidations all over her lung fields (Figure 2). Intravenous linezolid was added to her regimen empirically in order to treat a possible community-acquired Staphylococcus aureus pneumonia.

A repeat one-step RT-PCR in a nasal sample (Taqman, in-house protocol, Hellenic Pasteur Institute) confirmed the exclusive presence of human coronavirus 229E (HuCoV-229E). After the administration of systemic corticosteroids, the patient started to display clinical improvement within the first 24 hours. Further laboratory analyses did not reveal any immune defect. After a week, she was discharged from the hospital well and remained healthy 23 months later (Figure 3).