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Influenza is a common disease that causes annual epidemics, leading to medical, social and economic problems. Together with the elderly population, children under 5 years of age, even in the absence of underlying chronic diseases, have the highest risk of severe disease leading to hospitalization and, although rarely, to death. Influenza is estimated to be the cause of approximately 374,000 hospitalizations of children <1 year of age and 870,000 hospitalizations of children <5 years of age annually. Moreover, children are the most important cause of the spread of the infection in communities because they shed the virus in greater amounts and for longer periods of time than adults.
Many studies have demonstrated the risk of superinfection by S. pneumoniae and S. aureus during influenza, with a significant increase in the number of medical visits, drug prescriptions, and hospital admissions for respiratory disease. Influenza-related complications seem to be more common in children with underlying chronic severe diseases, which is why health authorities worldwide have long recommended that these children receive the influenza vaccine every year. However, recently collected data clearly demonstrate that otherwise healthy children can also suffer from severe influenza and that the annual number of deaths is not different from that in children with chronic severe diseases.
Every year during the influenza season, together with the two subtypes of influenza A virus (A/H1N1 and A/H3N2), two lineages of influenza B viruses (B/Victoria/2/87-like and B/Yamagata/16/88-like) simultaneously circulate; in some years, the influenza B viruses are responsible for the major disease burden. Unlike influenza A viruses, humans are the sole host with epidemiological relevance for influenza B viruses. Influenza B viruses evolve mainly through genetic reassortment between strains of different lineages. This allows for the escape from host immunity and the preservation of the ability to cause disease. Regardless of the lineage, influenza B infection can cause severe disease and death. During influenza season, influenza C virus can circulate infecting humans, dogs, and pigs, sometimes causing severe illness and local epidemics; however, influenza C virus is less common than the other types of influenza viruses and usually it only causes mild disease in children.
In the past, recommended vaccines included only one influenza B lineage, chosen by the World Health Organization (WHO) based on surveillance data regarding the lineage that had been observed to dominate in the previous year; however, currently, quadrivalent vaccines containing both influenza B lineages represent the best influenza prevention strategy. Studies have shown that the administration of quadrivalent inactivated (IIV) and live attenuated (LAIV) influenza vaccines to healthy children is effective at reducing the total burden of influenza, including preventing severe cases and saving costs due to productivity losses in parents and school absenteeism, with an acceptable level of safety and tolerability.
The USA Advisory Committee on Immunization Practices (ACIP) recommends influenza vaccination for the entire paediatric population, regardless of age and health conditions, starting from a minimum age of 6 months for IIV and a minimum age of 2 years for LAIV vaccines. The ACIP highlights the importance of administering 1 dose of any influenza vaccine annually to prevent influenza disease and complications, with 2 doses separated by at least 4 weeks for children 6 months–8 years who did not receive at least 2 doses of influenza vaccine before 1 July 2018. Within Europe, there are huge variations in influenza vaccine recommendations; for example, in Italy, as in some other countries, the influenza vaccine is recommended only for at-risk people and not healthy children.
The need to protect neonates and infants in the first 6 months of life from influenza is so important that many health authorities have recommended influenza vaccinations of pregnant women. Studies have shown that vaccination during pregnancy is effective in reducing influenza cases for at least one influenza season.
Experts agree that the available influenza vaccines can significantly reduce the risk of contracting influenza in healthy or immunocompromised subjects of any age. Preventing influenza infection through vaccination may decrease the subsequent burden of infection by some of the bacterial pathogens, reduce hospitalizations, and reduce antibiotic prescriptions for influenza complications in children and adolescents. Moreover, a reduction in influenza cases can have an impact on the abuse of antibiotics that are irrationally prescribed to a large number of paediatric patients with uncomplicated influenza, thus limiting the increase in antimicrobial resistance.
Bordetella pertussis is a Gram-negative coccobacillus that causes whooping cough and persistent cough, especially in neonates, school-aged children and adolescents.
The classic manifestation of the disease can be divided into three phases: (1) nonspecific symptoms, such as coryza, fever, and occasional cough; (2) constant and uncontrollable cough after two weeks, followed by forced inspiration producing a whooping sound; and (3) the convalescence phase, in which symptoms decrease progressively, and complications can appear. Complications, such as pneumonia, are frequent and are responsible for over 90% of the deaths attributable to the disease in children younger than one year of age.
There has been an increase in the incidence of pertussis in the European Union from 2011 onwards. The resurgence of pertussis observed in recent years seems to be a complex but real phenomenon due to a number of reasons, including the use of acellular pertussis (aP) vaccines in many locales. Lack of mucosal immune responses after aP vaccine administration favour infection, persistent colonization and transmission of the pathogen. Moreover, earlier waning of protective immunity and the circulation of B. pertussis variants depleted of vaccine-included antigens further favor the increase in pertussis disease. Studies in immunized children have reported that antibody responses and protective immunity wane 3–5 years after immunization with acellular pertussis (aP) vaccines, which may reflect poor induction of memory T and/or B cells.
The majority of health authorities recommend the administration of 2–3 aP vaccine doses in the first year of life with the administration of booster doses at pre-school age, during adolescence and then every 10 years during adulthood. Recently, in several countries vaccination against pertussis in the 2nd and 3rd vaccination during pregnancy is recommended in order to prevent pertussis in the first 6 months of life. Studies showed > 90% effectiveness of pertussis vaccination of mothers against pertussis in the first 6 months of life. Vaccine administration in pregnancy is safe for both mother and foetus.
In conclusion, prevention of pertussis with currently available vaccines reaching high vaccination coverage rates remains a priority, including the vaccination of pregnant women. Several different aP vaccines are available, but it has yet to be determined which of them confers the highest and the most-prolonged protection. Further studies are needed to evaluate the importance of individual antigens included in aP vaccines in conferring protection against disease, colonization, and transmission. However, present knowledge seems to indicate that pertussis toxin, particularly if genetically detoxified, represents the main antigen that ensures protection from disease even if not from infection. The optimal pertussis vaccine would be one that induces both mucosal and systemic responses similar to those occurring under natural infection, leading to long-term protection against both disease and infection. Such a vaccine might increase public confidence and result in better vaccine uptake.
Of a total of 65 clinically suspected cases, 21 infants (32.3%) were enrolled to be the confirmed cases of B. pertussis infection in this study. The age range was from 22 to 198 days old with a mean age of 2.5 months old. There were 11 males and 10 females. All were born full term at birth. Nine patients (42.9%, average age: 47.4 days) had not received any DTaP vaccinations, 9 (42.9%, average age: 77.2 days) received 1 dose, 2 (9.5%, average age: 104.5 days) received 2 doses, and 1 (4.8%, age: 198 days) received 3 doses of DTaP (Table 1). Pertussis occurred mostly in spring, summer and early fall, peaking in April, June, and September. There was no patient in the winter month from December to February (Fig. 1).
Although paroxysmal cough manifested for more than 1 week in all cases, whooping cough was present in only 3 cases (14.3%). There were 4 cases (19.0%) of pneumonic infiltration, 3 cases (14.3%) of hyperinflation on radiologic examinations. Four patients with abnormal chest radiographic finding were not immunized with DTaP vaccination, and remaining 3 patients were received with 1 dose of DTaP vaccination. All patients except 2 cases were examined by multiplex PCR for respiratory virus identification. Two patients had concomitant infections of rhinovirus and respiratory syncytial virus, respectively, and severity was not increased in these patients. Absolute lymphocytosis was noted in 12 patients (57.1%). Of note, absolute lymphocytosis was found more commonly in patients (8/9, 88.9%, average age: 45.8 days) who did not receive DTaP vaccination than patients (4/12, 33.3%, average age: 88.0 days) who did receive DTaP vaccination (P = 0.024, Table 2). The mean length of hospital stay among all patients was 11.7 days. However, the mean length of hospitalization for patients without DTaP vaccination was 15.4 ± 6.6 days, which was significantly longer than the 8.8 ± 3.8 days of patients with DTaP vaccination (P = 0.009, Table 2). Two patients were admitted to the intensive care unit (ICU); both of them did not receive DTaP vaccination, and one of these patients treated with a mechanical ventilator. There were no deaths due to pertussis during the study period (Table 1).
On the result of diagnostic methods, PCR had the highest sensitivity showing positivity for all 21 cases (100%), however, confirmed cases with culture were 9 (42.9%) which showed the lowest sensitivity. Although RT-PCR and serological test were not performed in all patients, high sensitivity was observed in all patients done with these tests.
Total of 72 family members and long-term guardians of index infant cases were enrolled in this study for confirming household transmission. The composition of those members were as followings; 42 parents, 20 siblings, 10 relatives. Seventy members (97.2%) showed respiratory symptoms, and 42 members (60%) with respiratory symptoms had a history of antibiotics use. Of these 72 family members, 38 members (38/72, 52.8%) were confirmed with laboratory criteria of pertussis as followings; all 38 members were positive by PCR, 31 members (who received ELISA assay) were all positive serology, 12 members were positive culture. Finally, a total of 18 potential family sources (18/21, 85.7%) were identified for confirmed cases, of which 20 members (20/38, 52.6%, 15 family) were parents and 8 (8/38, 21.1%, 6 family) were siblings. Other 10 members (6 family; 6 grandparents, 4 aunts) accounted for 26.3% (10/38). In particular, mothers provided the highest proportion (13/38, 34.2%) of presumed sources of infection (Fig. 2).
In conclusion, about one third of infants with B. pertussis may have a respiratory viral coinfection. These undetected coinfections seem to leave the clinical severity of pertussis in infants unchanged.
In this descriptive study investigating the clinical data for 53 infants younger than 180 days hospitalized with pertussis, no associations between clinical severity and pertussis with or without co-infections were found.
As many as 25/53 (47%) of the infants hospitalized with pertussis over the two years were coinfected with other respiratory viruses. A distinctive point is that because we analyzed a wide virus battery and did so in infants hospitalized continuously in two centers over two years we feel confident that our study provides reliable data on the distribution of respiratory infections. Few studies found B. pertussis cases in infants with respiratory pathogens. For example, Piedra et al., identified by RT-PCR only 4 B. pertussis cases in 2068 patients with respiratory pathogens and all these infants were younger than 6 months. In 3 of the 4 infants with B. pertussis RT-PCR identified a second respiratory pathogen: 2 had an hRV and 1 child an hCoV coinfection. In a similar study, Korppi et al., showed in a naso-pharyngeal aspirate by RT-PCR B. pertussis infection in 7 on 9 of patients hospitalized for RSV infection. Our lower percentage of coinfections reflects the analysis of a larger series of children with B. pertussis than the other two studies.
As well as extending current epidemiological knowledge on respiratory pathogens, we confirmed that pertussis in Italy arises mainly during the summer months, whereas respiratory virus coinfections are equally distributed over the year. Virological analysis showed that the most frequent virus observed was hRV (36%) and only 2 infants had RSV (8%). We presume that the distribution of coinfections reflects the observation that hRV epidemiology maintains a steady epidemic curve throughout the year and hRV can be detected also in asymptomatic children. In fact in a Netherlands study conducted in recent years Wildenbeest JG et al., found hRV in 25% of asymptomatic children.
A new finding in this study is the lack of differences in clinical disease severity (measured as clinical severity score and days hospitalization) in infants with B. pertussis infection alone and those with coinfections. Although no other published studies have tested 14 respiratory viruses in infants with pertussis, Nuolivirta et al., studied 142 infants aged less than 180 days hospitalized for bronchiolitis who underwent a nasopharyngeal aspirate to detect only 7 respiratory viruses and B. pertussis by RT-PCR. B. Pertussis involvement was found in 12 of 142 (8.5%) infants hospitalized for bronchiolitis and, of these, 8 were in coinfection with RSV. They found no differences in clinical findings, days hospitalization and breastfeeding at admission in patients with respiratory viruses alone than in patients with respiratory viruses in coinfections with B. pertussis. Schnoeller et al. showed that respiratory infection of neonatal mice with an attenuated B. pertussis can protect against RSV-induced disease in adult life [18, 19]. In a human model Schiavoni et al. demonstrated that an attenuated B. pertussis rescues the immune functions of RSV infected human dendritic cells by promoting a protective Th1/Th17 responses. Self-limiting respiratory infections with attenuated bacteria or commensal microbes may have a beneficial effect limiting potentially lethal diseases caused by respiratory viruses in infants.
When we evaluated the secondary endpoints in our study, infants with B. pertussis alone were younger, less often breastfeed and breastfeed for a shorter time than infants with coinfections. Similarly, in a previous study from our group, comparing infants with B. pertussis and those with bronchiolitis, we found that the percentage of breastfed infants at hospitalization was lower in infants with B. pertussis than in those with RSV bronchiolitis. This finding is difficult to explain. We always strongly encourage breastfeeding in infants because it has well-known immunological and nutritional advantages. In a recent large multicenter study, on pertussis-associated pneumonia in children from low- and middle-income countries, pertussis-positive cases were more likely to have never been breastfed compared with controls. Our finding might reflect mother-to-child transmission of respiratory infections during breastfeeding.
Our study has limitations: small sample size and our failure to compare B. pertussis infection with viral coinfections by single respiratory viruses.
A resurgence of reported pertussis has been documented in a number of countries with high vaccine coverage since the 1990s (1, 4, 18). DTaP vaccination coverage in Korea is very high, with 94% of the children completing the primary series of pertussis immunization. Despite the nationwide vaccination program with high coverage, the incidence of reported pertussis has continued to increase from 2000. Understanding the epidemiological and clinical features of pertussis in community is important because it remains a major cause of morbidity and mortality worldwide. In this study, we have made the demographic data from confirmed pertussis in an attempt to compare the clinical outcomes according to DTaP immunization. Generally, pertussis mostly occurred in young infants under 6 months of age who did not complete the primary series of DTaP vaccination. Depending on the immunization status of our patients, there were significant differences in clinical outcome similar to other studies (11, 19, 20). The duration of hospital stay was significantly longer; and the severe cases admitted to ICU were found in patients without pertussis vaccination as compared to those with vaccination. The absolute lymphocytosis in laboratory findings was shown primarily in patients without the vaccination history. The main presenting symptom in typical pertussis is whooping cough. Even though all patients had paroxysmal cough for more than 1 week, whooping cough was present in only 14.3% of the patients. In addition, most patients showed no specific findings in chest radiographs. Because the presenting symptoms may be nonspecific, it is important for clinicians to suspect pertussis when there is prolonged cough or absolute lymphocytosis, especially in young infants with no or incomplete DTaP vaccination.
The highest numbers of pertussis cases was in April, June, and September, which have relatively warm temperatures in Korea. Few patients occurred in winter as other respiratory pathogens are commonly considered in the differential diagnosis. Similar studies from other countries have also shown that the reported pertussis case mostly occurred in a summer months with high temperatures (21, 22). Since the number of patients is too small, it is unclear to know the seasonality of pertussis based only upon this result. In addition, various factors such as environmental factors, school opening, and climate can affect the seasonal characteristics of pertussis epidemiology. However, because clinical presentations of pertussis often resemble influenza-like symptoms and those of other respiratory diseases such as Mycoplasma pneumoniae and adenovirus, this lack of awareness and nonspecific clinical characteristics may be often associated with underdiagnosed condition of pertussis.
The diagnostic tool of pertussis is challenging. Culture of nasopharyngeal secretions is essential for diagnosis, however, the result showed the lowest sensitivity (42.9%) in our patients. Moreover, this result requires a long incubation period up to 10 to 14 days, after which the patient can be delayed in critical treatment and infect other persons with contact. The majority of confirmed cases were based in the presence of PCR. Although RT-PCR and serological test were not performed in all patients, these methods also have higher sensitivity than culture. Serologic testing can help the diagnosis of patients with atypical symptoms, for whom clinical samples are collected at later time from the onset of disease.
This study is, to our knowledge, the first to document the presumed importance of household transmission in Korea. In 18 families (85.7%) of our patients where a source of infection was established, parents (52.6%), grandparents and aunts (26.3%), or siblings (21.1%) were identified as the frequent source. In particular, mothers provided the highest proportions of presumed sources of infection.
In our study, the majority of household members showed respiratory symptoms such as prolonged cough and recent history of antibiotics treatment for respiratory infections. However, none of them did know that pertussis might cause severe or chronic cough in adolescents and adults. Also, they did not have the concept of household transmission of pertussis. There has been an increase in the reported number of pertussiss cases in many countries, primarily among adolescents and adults, which is special concern because adolescents and young adults are a recognized reservoir of infection for neonates and infants, who are at high risk for pertussis-related morbidity and mortality (23-26). Several household studies have noted the predominant role of parents in the transmission of pertussis to susceptible infants (27, 28). For this reason, the USA Advisory Committee on Immunization Practices (ACIP) has recommended the cocooning strategy that Tdap vaccine be administered to all caregivers of infants less than 1 yr, in an effort to protect young infants against pertussis since 2006 (29). Likewise, the cocooning strategy is important in Korea, in keeping with high morbidity and mortality in young infants without immunization (30). Despite the introduction of Tdap vaccine in our country, vaccine use is only beginning, perhaps because of an incomplete understanding of the burden of pertussis among community population and the lack of national policy and education concerned with the necessity of Tdap vaccination. The immunization policy in Korea recently introduced the booster dose of Tdap vaccine as part of routine immunization in adolescent. Therefore, the change in the epidemiology of pertussis should be noted carefully.
In conclusion, pertussis is still present in Korea and remains a major morbidity in young infants. Most infants were too young to have received the primary series of DTaP vaccination before infection. Household members were identified as a potential source of pertussis infection, and they should be encouraged to receive booster immunization with Tdap to minimize pertussis transmission to this vulnerable group on infants. Nationwide pertussis reporting is also urgently required to better understand disease transmission patterns, for early recognition and prevention of outbreak and for the evaluation of vaccination policy. Universal recommendation for pertussis booster vaccination at 11 to 12 yr is expected to decrease the transmission of pertussis in households in Korea. The surveillance of pertussis outbreak should be continued for controlling this disease.
Data on age (Table 2) and gender were available for all 413 patients seen for ILI in both hospitals.
Overall, 124 of the 413 patients (30.0%) were less than 15 years old (4 in SLS and 120 in TRS) and 281 patients (68.0%) were under 40 years of age (68 in SLS and 213 in TRS). In SLS, the median population age was 41 (Interquartile range [IQR]: 28–56) with 49.7% being males, whereas in TRS, the median population age was 17 ([IQR = 3–34]) with 51.1% being males.
In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen (Table 2). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4). However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years (Table 2). PIV infection occurred in very young patients (median age = 4 vs. 29 for patients without PIV, p<10−4) (Figure 4). The same observation was made for ADV infection (median age = 2.5 vs. 25 for patients without ADV, p = 0.006) (Figure 4). Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p<10−4 and p<10−3 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4), influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2–18.5 vs. 25, 6–43) and rates of mixed infection were significantly higher in patients under 15 years as compared to older ones (19.4% vs. 4.1%, p<0.0001).
At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379).
Overall, most patients have fever (93.9%) and cough (86.1%) (Table 3). Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39°C; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown).
In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) (Table 3). Similar median temperature was reported in H1N1v positive and in H1N1v negative patients (39 [IQR = 35.5–41] vs. 38.8 [IQR = 37.8–40.4], p = 0.68). The proportion of patients with a temperature above 39°C was not different (H1N1v positive: 34.3% vs. H1N1v negative: 32.3%, p = 0.84) (data not shown).
We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFinder19®) (Table 3). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens (Table 3).
As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively).
Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39°C, which was not different from patients with single viral infection (28.6%).
Between October 2011 and March 2014, a total of 432 hospitalized children with ARI, most of them with community-acquired pneumonia, influenza-like illness, or asthma exacerbation, were enrolled in this study (Table1). The median age of study participants was 36 months, and 51·4% participants were male.
At least one respiratory virus was detected in 258 (59·7%) of NPS from the enrolled patients. The most common were HRV in 115 (26·6%) patients, followed by RSV (17·4 %), influenza A (8·6%), HMPV (3·5%), HPIV (3·5%, with HPIV-3 being the most frequent), ADV (2·8%), and other viruses less frequent as bocavirus and HCoV with eight and seven infections detected, respectively. Single viral infections were detected in 183 samples (42·4%), and two or more viral infections were found in 21 samples (4·9%), where the most common coinfections were those involving HRV and RSV with six cases, HRV and HCoV in three samples, HRV and ADV or influenza A, with two cases each one. Coinfections between HRV and bocavirus or HPIV, RSV-A were detected once each one, and also RSV-B and ADV, bocavirus, metapneumovirus, HCoV and HPIV were detected once each one. A total of 36 samples (8·3%) had only bacterial infection, where Haemophilus influenzae was the most common with 17 cases, and other bacteria detected were Streptococcus pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis, and Chlamydophila pneumoniae. Viral and bacterial coinfections were detected in 53 samples (12·3%), with the coinfection between HRV/H. influenzae and HRV/S. pneumoniae as well as RSV/H. influenza the most frequent. A total of 139 samples (32·2%) did not have any respiratory pathogen (Table2).
In conclusions, clinical manifestations of pertussis in infants can overlap with several different diseases. Sometimes presentation may mimic a viral respiratory tract infection. Our data support a routinely use of RT-PCR for pertussis in all infants ≤3 months of age with any respiratory symptoms in order to implement appropriate control measures in hospital and in the community at large as the clinical suspicion is often not enough to well recognize pertussis infection.
For viruses, the overall prevalence was higher in younger patients (Table 4, p = 0.046). Rhinovirus was the most prevalent cause of infection in <40-year-old patients (34.4%) and showed a trend of declining prevalence with older age (p = 0.044). For typical bacterial agents, there was a trend of higher prevalence at older ages (p = 0.001). There were no significant differences for atypical pathogen. The prevalence of co-infection and mixed infection did not differ across the three age groups.
We compared clinical characteristics according to microorganism groups (Table 5). The bacteria-only group was significantly older (mean age, 52.2 vs. 43.3 years; p = 0.004) and included significantly more men (52.0% vs. 34.0%; p = 0.037) than the virus-only group. Only sore throat was more common in the virus-only group than in the bacteria-only group (32.7% vs. 52.0%; p = 0.023). Patients with mixed infections (55/291) showed intermediate values between the bacteria-only and virus-only groups in terms of mean age, sex ratio, and sore throat prevalence.
ARIs as the most common cause of morbidity and mortality in children, remaining a major concern, especially affecting children under 5 years old from low-income countries [1–4]. Unfortunately, information regarding their epidemiology is still limited in Peru [7, 11].
In recent years, there has been evidence of pertussis resurgence in Latin America, despite the introduction of the vaccine [14, 27, 28]. This bacterium is highly contagious and virulent, about half of the infected children under one year of whooping cough require hospitalization. In Peru, the national immunization program administers the combined pentavalent vaccine (DPT, HvB, Hib) at 2, 4, 6 months with reinforcements at 18 months and 4 years [29, 30]. Thus, we conducted a previous study on Peruvian children with a probable diagnosis of Pertussis and reported a Bordetella pertussis prevalence of 39.5%. However, the classical presentation of pertussis has proven to be not enough to achieve a definitive diagnosis and laboratory tests are of the utmost importance for etiological confirmation to avoid overdiagnosis [28, 31, 32]. In the light of possible coinfections and more than 60% of patients without an etiological identification in our previous study, we conducted a new comprehensive analysis to detect viral and atypical bacterial etiologies in all our patients.
From a total of 288 samples analyzed from children under 5 years old with a probable diagnosis of Pertussis, the most common pathogen isolated was ADV in 49% of samples, followed by B. pertussis in 41% from our previous analysis. Although this study was conducted in patients with ARI with a highly suspicious pertussis diagnosis, other studies on children with ARI have identified ADV as one of the most prevalent etiologies Although, the viral and bacterial prevalence may vary widely depending on the population characteristics [8, 33, 34].
Interestingly, our population were children with a highly suspicious clinical diagnosis of pertussis and despite that whooping was present in 46.5% of patients, ADV was the most common etiology isolated. Furthermore, a great number of patients with ADV infection presented with clinical symptoms very common among patients with pertussis such as whooping (51.1%), shortness of breath (43.4%) and vomiting (47.5%). Similarly, a recent study has reported that gastrointestinal symptoms and difficulty breathing are among the most common type of presentations in children. Additionally, ADV has been historically identified as a major cause of pertussis-like syndrome, which results in the likelihood of a pertussis misdiagnosis in the absence of laboratory confirmation [36–38]. In this way, we have shown that patients with infection by ADV and B.pertussis as a single infectious agent have similar symptoms (Fig. 1).
It is also important to highlight the presence of Mycoplasma pneumoniae (26%) and Chlamydia pneumoniae (17.7%) among our patients. In a previous study, in children with ARIs, we reported a very similar prevalence of Mycoplasma pneumoniae and Chlamydia pneumoniae in 25.2 and 10.5%, respectively. Demonstrating the high prevalence of these atypical bacteria among Peruvian children with ARIs. Our results from this current study also make noteworthy that clinical manifestations by Mycoplasma pneumoniae and Flu-B, ADV, or B. pertussis are distinguishable when the infection is due to infectious agent alone. However, in coinfections the symptoms were undestinguibles (see Table S4).
Additionally, in our previous study coinfections between these bacteria and viruses were also frequent; present in 67.4% of samples, coinfections between were the most common combination and the association between Mycoplasma pneumoniae with VRS-A was the most frequent one observed in 9.59% of patients. Surprisingly, in this study, we have observed 58% of coinfections in our samples, again being the viral-bacterial association the most frequent and the most commonly detected coinfection involving Bordetella pertussis-ADV and Mycoplasma pneumoniae-ADV with frequencies of 12.2 and 6.5%, respectively. Another study in children, although in patients with community-acquired Pneumonia, also have reported coinfections in M. pneumoniae as the most common bacteria detected in association with a virus. Thus, to avoid under-diagnosis, pertussis should be considered in patients with cough, especially if chronic, even when M. pneumoniae have been documented.
Another common coinfection was B. pertussis and Flu-B present in 9 patients. Although viral-bacterial coinfections are commonly associated with worse clinical courses and longer hospitalizations [17, 19, 20]. Recent investigations have reported similar clinical outcomes in infants hospitalized with B. pertussis and another respiratory virus coinfection. However, noteworthy attention should be given to the B. pertussis and ADV coinfection in infants. A study compared infants with RSV and RSV-B. pertussis coinfection reporting similar disease severity; however, patients with this coinfection clearly needed more respiratory care and nutritional support. Consequently, our only patient with RSV-A and pertussis presented with cyanosis and required advance respiratory support.
The variations in the rate and pattern of coinfection in patients with ARIs may be related to seasonal and geographical factors. In our study, we intended to describe all detected pathogens and their seasonal distribution. Even though we were not able to describe any clear pattern, it is worth mentioning the high prevalence of ADV and M. pneumoniae across all of the study period, as well as the increasing prevalence of B. Pertussis on 2012.
Young infants are at high risk of critical pertussis. Despite advances in life support and the treatment of organ failure in childhood critical illness, critical pertussis remains difficult to treat.
In a retrospective single-center study, from a group of infants hospitalized from October 2008 to April 2010 at our Pediatric Emergency Department for acute respiratory symptoms we selected for study 19 consecutive infants aged less than 12 months (6 boys, median age 72 days, range 20-187) with Real Time-PCR confirmed pertussis. We also analyzed data for B. pertussis variants among hospitalized patients in whom B. pertussis was cultured. As a control group, we recruited 19 age- and sex-matched infants (6 boys, median age 71 days, range 20-183) from 164 infants, hospitalized during the same period with RT-PCR confirmed RSV bronchiolitis and negative for B. pertussis. The diagnosis of bronchiolitis was considered in infants less than 12 months with the first episode of acute infection of the lower respiratory tract. Infants with co-morbidity were excluded.
Detailed demographic, clinical and laboratory data were obtained from patients’ parents with a structured questionnaire and from medical files. Clinical outcome variables evaluated included gender, gestational age, birth weight, type of delivery, DTaP (diphtheria, tetanus and acellular pertussis) vaccination received, breast feeding history, age and weight at admission, number of siblings, siblings’ schooling, cough at admission (presence and duration), paroxysmal cough, presence of fever (body temperature >37.5°C), apnea and cyanosis, chest sounds, and hospitalization days. Laboratory outcome variables investigated were white blood-cell count (WBC), lymphocyte count, eosinophil count, neutrophil count, platelet count, hemoglobin (Hb), glutamic oxaloacetic transaminase (SGOT), glutamic pyruvic transaminase (SGPT), gamma-glutamyl transferase (GGT) and C-reactive protein (CRP). At hospital admission, each infant was assigned a clinical severity score ranging from 0 to 8, according to respiratory rate (<45/min = 0, 45-60/min = 1, >60/min = 2), arterial oxygen saturation in room air (> 95% = 0, 95-90% = 1, < 90% = 2), retractions (none = 0, present = 1, present + nasal flare = 2), and ability to feed (normal = 0, reduced = 1, intravenous fluid replacement =2).
All infants’ parents were asked to participate in the study and gave written informed consent. The study was approved by the Policlinico Umberto I institutional review board (Reference n° 2377/09.02.2012).
The clinical suspicion of pertussis is not easy in infants. Among the 53 patients BP+, pertussis was suspected only in 16 patients (30.2 %) at the admission. Clinical suspicion has a low sensitivity in this age group. In absence of a systematic use of laboratory tests for the diagnosis of pertussis in infants with respiratory symptoms, many infants with BP infection may go unrecognized. In a hospital environment, the lack of recognition of such an infection may represent a severe risk for hospital outbreaks, since pertussis may be transmitted to contacts if appropriate antibiotic therapy is not applied. As a matter of fact we did not observe any hospital secondary pertussis case over the study period.
Our data suggest that, despite routine surveillance shows a low incidence of pertussis in Italy, BP infection is still circulating in unvaccinated infants and nearly 25 % of patients younger than 3 months of age hospitalized with respiratory symptoms resulted positive for BP in our case series, according to what previously reported. Clinical manifestations of pertussis can overlap with those of other diseases and can be atypical. Nonetheless our data, in line with the literature [19–21], suggest that some feature should alert clinicians to suspect pertussis: the hallmark clinical characteristic is paroxysmal cough; fever is usually absent and laboratory findings showed marked lymphocytosis. On the other hand the absence of typical symptoms does not exclude the diagnosis of pertussis.
Furthermore, it has been demonstrated that vaccinated children and adolescents could have more silent or mild pertussis infections [22, 23]; so we might expect that infants born to mothers who had received Tdap during pregnancy could present a modified pertussis. In our series, no infants were born to mothers who had received a booster during pregnancy.
Of note, eight of our patients (15.1 %) with confirmed BP infection were admitted with diagnosis of apnea, not associated with other symptoms, according to what previously reported by Crowcroft and Shojaei [24, 25]. In the ECDC pertussis case definition of 2012, apnoeic episodes in infants are included in the clinical criteria.
The lack of recognition of BP infection in infants has important consequences for surveillance. The number of infections that our hospital detected in the study period does not seem consistent with national surveillance data that suggest a low circulation of pertussis in the first year of life. Although we may have experienced an increased incidence limited to our geographic area, we speculate that other health facilities that do not systematically apply laboratory confirmation to all infants with respiratory symptoms may miss cases as well.
We did not observe deaths in our case series. Since cases included in our study were admitted in a general pediatrics ward we cannot exclude that other severe cases were admitted to pediatric intensive care unit (PICU). Crowcroft et al. in a previously study, described that about 20 % of infants <5 months admitted to London PICU, with respiratory failure, apnea, or acute life threatening episode had pertussis. They reported two cases of death.
One of the barriers to wide use of RT-PCR for pertussis may be costs associated with laboratory tests. Beyond the tremendous impact of early therapy in pertussis cases, it should be underlined that RT-PCR for pertussis has an additional cost of nearly 30 euros or 38 USD per test.
The use of molecular assays has become standard of care in many setting recently. Mainly PCR has the advantage of offering result within several hours and in most of the cases, it is considerated highly sensitive especially during the “late stage” of the disease. Moreover, because is not affected by the use of drugs, one of the major usefulness respect to cultures methods is that specimens could be collected after antibiotic treatment has been started. Phenotypic cultures, although highly specific, fall in sensitivity with a reported range between 30 and 60 % of BP detection and increased time for releasing test result compared with PCR. We account as great limitation of PCR that it’s unable to differentiate asymptomatic carriers. In our series, all the patients were admitted for acute respiratory symptoms and were unvaccinated so we considered them as affected by pertussis and to be properly treated.
The PCR target used for diagnosis in this study was IS481, consequently we cannot distinguish between BP, Bordetella holmesii and Bordetella bronchiseptica. Therefore, we consider it is unlikely that our data are a possible overestimation of true data.
The IS481 sequence is represented from 50 up to 238 copies per cel in BP considering this an advantage in term of high sensitive detection. Notwithstanding, microbiologist, infectious disease practitioners and pediatrics should be acquainted with the low presence of this target in Bordetella holmesii (8 to 10 copies per genome) and Bordetella bronchiseptica as well [28–30]. Some laboratories perform a second PCR assay on IS481-positive specimens using either a BP- or a Bordetella holmesii-specific target or both. However, these other targets are at least ten fold less sensitive than IS481 target since they are present in fewer copies of the genome. A number of potential target sequences have been proposed to increase the diagnosis of BP and Bordetella holmesii but there is no recommendation on what is the best PCR diagnosis strategy to use at the present time.
We recommend referring the final species differentiation of pertussis illness depending on several factors as by the setting of patients (more often related to immunocompromised individuals), or being contingent on epidemiology contest.
Regarding Bordetella parapertusiss, in many clinical laboratories due to the high prevalence of pertussis in order to better exploit the species responsible of pertussis disease, it is suggested to investigate at the same time RT-PCR assays by using IS481 for BP and IS 1001 specific of Bordetella parapertussis. These double check methods could represent an appropriate procedure in order to estimate the prevalence of Bordetella pertussis/parapertussis infections, and to determinate their epidemiologic characteristics in infants.
Although the duration of symptoms in BP+ patients was longer than in the other diagnoses, 40 patients (75.5 %) of 53 BP+ showed cough for less than 14 days. According to the clinical case definition of CDC, WHO and ECDC, none of those patients should have been reported. Similar observations were reported also by others [25, 34]. The case definitions and classifications played an important role in improving the sensitivity of pertussis diagnosis in epidemiological surveys. But if these criteria are used purely clinically to select pertussis cases for confirmation in this age group, this may result in considerable under-diagnosis of pertussis. The clinical suspicion does not always meet all the clinical criteria. Therefore, the current clinical criteria of pertussis are all based on paroxysmal cough, but as van den Brink et al have previously demonstrated, it is not a good predictor in atypical pertussis infections. The development of an age-related case definition, as suggested by the GPI, may be important to better assess the real burden of pertussis in infants.
Our data support what has been previously reported by Gonfiantini et al. about the pertussis seasonality. Many patients were affected by pertussis during summer period; nevertheless some cases were reported even in winter when most of respiratory viruses circulate among population.
BP and RV co-infection was documented in 34 % of our subjects. Conflicting results have been reported regarding the frequency of these concomitant [34–37]. The short duration of surveillance, the inclusion of highly selected patient groups based on age and diagnosis may have contributed to these heterogeneous findings. Therefore, in most of the previous studies, has been taken into account only the co-infection between RSV and BP. In our study we analyzed co-infections between BP and the most frequent RV.
From the clinical point of view, it’s important to recognize co-infections, both for infection control and clinical management. A diagnosis of respiratory virus infection does not exclude pertussis and viceversa.
This study has some limitations. The retrospective data collection from medical records can contain inaccuracies regarding clinical information, but objective parameters were analyzed in order to reduce the possibility of bias. Second, we cannot say if our results may be generalizable to other pediatric settings in our country. Nonetheless the use of laboratory test for pertussis confirmation is not widespread in Italy and, despite EU recommendations for case definition, most reported cases are still diagnosed on a clinical base only. Lastly nineteen of our patients were treated with macrolides before admission; this may have mitigated the clinical manifestations in some cases.
Several strategies have been proposed for prevention of pertussis in infants. These include immunization of adolescents and adults with tetanus, diphtheria toxoid and acellular pertussis (Tdap) to boost waning immunity against pertussis. Recent study, however, demonstrated that after Tdap introduction in adolescents, the incidence in this age group was reduced but the average incidence of pertussis among infants younger than 1 year did not significantly change. This is probably because of increased vaccination coverage of those patients at the highest risk to transmit disease is needed before the indirect effects of Tdap are fully realized [39, 40].
Another proposed strategy is maternal immunization during the third trimester of pregnancy; from October 2011, the Advisory Committee on Immunization Practices (ACIP) recommended that unvaccinated pregnant women receive a dose of Tdap. Vaccination of women with Tdap during pregnancy is expected to provide protection to infants from pertussis until they are old enough to be vaccinated themselves. Tdap given to pregnant women stimulate the development of maternal antipertussis antibodies, which pass through the placenta, likely providing the newborn with protection against pertussis in early life, and protect the mother from pertussis around the time of delivery, making her less likely to become infected and transmit pertussis to her infant. In England an immunization program against pertussis for pregnant women was introduced in October 2012 in response to a marked increase in pertussis cases, particularly in young infants. The program achieved 60 % vaccine coverage and 90 % vaccine effectiveness in preventing infant disease was demonstrated.
Vaccination of pregnant women is considered likely to be the most cost-effective complementary strategy to prevent pertussis-associated infant mortality.
Moreover, postpartum maternal immunization with Tdap, as well as immunization of close household members to prevent transmission of infection to vulnerable infants (cocooning strategy) is currently recommended by the Advisory Committee for Immunization Practices to the Center of Disease Control and Prevention. Since the impact of these strategies cannot be accurately monitored without reliable surveillance data, we believe that considering Bordetella pertussis infections in infants ≤3 months of age hospitalized with respiratory symptoms is essential.
A total of 288 patients under 5 years old with a probable diagnosis of B. ertussis were studied thouroughly for specific etiological identification. More than 80% of our study population were infants between 1 to 5 months old with a slightly higher number of males (56.3%). The group of infants between 29 days – 2 months-old (27.4%) and the group between 3 and 5 months-old (27.4%) were the most predominant, closely followed by the group between 2 and 3 months-old (26.4%) (Additional file 1: Table S1).
From our previous study, 118 cases of Bordetella pertussis were confirmed via PCR, leaving potentially 59% of samples without etiological identification. Thus, all 288 were analyzed for the presence of Influenza-A (Flu-A), Influenza-B (Flu-B), RSV-A, RSV-B, Adenovirus (ADV), Parainfluenza 1 virus (PIV-1), Parainfluenza 2 virus (PIV-2), Parainfluenza 3 virus (PIV-3), Mycoplasma pneumoniae and Chlamydia pneumoniae. The most common pathogen isolated was Adenovirus at 49% (141/288), followed by Bordetella pertussis at 41% (118/288), Mycoplasma pneumonia at 26% (75/288) and Flu-B at 19.8% (57/288) (Additional file 1: Table S1-A). The identification of these infectious agents has made it possible to establish remarkably that coinfections were present at 58% (167/288) of patients. Thus, cases of infection due to a single infectious agent were 28.8% (80/288), and where the presence of ADV was 10.2% (25/80) and for B. pertussis was 9.8% (24/80), followed by M. pneumoniae with 6.1% (15/80). Furthermore, the prevalence of these infectious agents were accumulated in children under 5 months of age (Additional file 1: Table S1-B).
As indicated above, in infected children (247 cases) coinfections stand out considerably (Additional file 2: Table S2). The coinfections found involve 2 to 6 different infectious agents, being the most frequent the coinfections of 2 agents with 39.6% (97/247) and those involving 3 agents with frequency of 23.2% (57/247). The most frequent association was the bacterial-viral coinfection, and the combination between Bordetella-ADV and Mycoplasma-ADV were the most common involvement reported with 12.2% (30/247) y 6.5% (16/247), respectively. However, the Bordetella-Mycoplasma association was very reduced (Additional file 2: Table S2), In addition, it is interesting to note that the associations in coinfections increase the frequency of infectious agents such as Chlamydia, Flu-B and RSV-A as observed in Additional file 1: Table S1 (compare 1A with 1B).
Regarding vaccination status, 65.25% (77/118) of the positive cases were unvaccinated. However, the majority of these children (40/77) were under two months of age. An unknown vaccinated status was observed in 5.93% (7/118) of patients positive for B. pertussis. A marked decrease was observed in children who had received at least one dose of vaccination, with a prevalence of 18.64% (22/118) (Additional file 3: Table S3).
In our population, the most common clinical symptoms registered at admission were vomiting (47.2%), whooping (46.5%) and shortness of breath (43.1%), followed by fever (35.4%) and cyanosis (28.8%). A wide spread distribution of symptoms distribution was observed when patients symptoms were individually assessed based on etiological group. Only 6 pathogens had symptoms that were present in more than 50% of each group. For example, vomiting was more commonly reported among children with Flu-A, RSV-A, Parainfluenza-1 and B. pertussis (Additional file 4: Table S4-A). However, the difficulty in establishing clear clinical symptoms associated with infectious causal agents is due to the high frequency of coinfections. Therefore, Additional file 4: Table S4-B has recorded the clinical symptoms of cases of infection with a single agent, and the association of these clinical symptoms are shown in Fig. 2. Thus, a clear non-association can be observed between Mycoplasma and Flu-B, ADV or Bordetella; the same happens for Flu-B and Bordetella or ADV. This non-association means that the only infectious agent could be identified taking into account the clinical symptoms of the children as shown in Additional file 4: Table S4-B.
The most common complications were acute bronchial obstructive syndrome (ABOS) and pneumonia in 56.6 and 28.1% of our population respectively. ABOS was the most frequent complications among patients with positive samples for RSV-A, Flu-A, ADV, M. pneumoniae, C. pneumoniae and B. pertussis. (Additional file 5: Table S5-A). However, when the complications of children affected by a single infectious agent are analyzed, it is clearly demonstrated that ABOS is also a complication of Flu-B. In addition, it is noteworthy that ABOS occurs in 61% (25/41) of the negative cases (Additional file 5: Table S5-B).
Finally, a seasonal distribution was described for each specific microorganism. Positive samples for ADV and Mycoplasma pneumoniae were observed across the whole study period. On the contrary, most of the B. pertussis cases were detected from May 2011 to March 2012. RSV-A and Chlamydia were mostly detected from March to May 2010; however, the same distribution was not observed in the following year (Fig. 3).
This is the first comprehensive detailed study of the diversity of respiratory microbes detected in children presenting with suspected PTB in a TB endemic setting and showed that multiple potential pathogens are present in th nasopharynx of such children.
The FTD33 multiplex real-time PCR detected at least one of the 33 microbial targets in 97 % of NP swabs from children suspected of PTB. Detection of multiple bacterial and viral targets was common. Bacterial species frequently found as commensals in the nasopharynx were most commonly detected [17, 18]. They include M. catarrhalis (64 %), S. pneumoniae (42 %), H. influenzae spp (29 %), and S. aureus (22 %). In addition, potential pathogenic organisms were detected in the nasopharynx including RSV, C. pneumoniae and B. pertussis.
The prevalence of B. pertussis was 6 % in our study. Similar detection rates (1–9 %) were reported in other South African settings in children with LRTI including at our study site, 10–20 years post transition from whole-cell vaccines to acellular vaccines (South African infants are vaccinated with DTaP-IPV/HIB; Pentaxime®, Sanofi Pasteur).
The prevalence of some clinically relevant viral targets (RSV, bocavirus and adenovirus) in this cohort is lower than that previously reported in children with LRTI [20–22]. The observed differences may be explained by our enrolment criteria which targeted symptoms suggestive of PTB. However, viral PCR positivity of hMPV, enterovirus and influenza virus is similar to a recent case-control study that also showed their association with community acquired pneumonia. As with bacteria, care needs to be taken with the interpretation of molecular detection of some viruses in NP specimens, since target nucleic acid may be detected for some time after resolution of symptoms, and from otherwise healthy children [23, 24].
In this study, some microbes showed no association with any of the TB categories. These included M. catarrhalis, and S. pneumoniae. A randomised controlled trial of the efficacy of PCV9 in South African children showed decreased rates of culture-confirmed and clinically diagnosed TB in PCV9 recipients hospitalised with LRTI compared with placebo recipients (relative risk reduction 43 %). This suggests that coinfection with M. tuberculosis and S. pneumoniae causes severe infection requiring hospitalization. Although common in our cohort, S. pneumoniae did not cluster together with the TB or unlikely-TB groups, however we measured NP colonization which is likely to be an inaccurate measure for the contribution of S. pneumoniae to LRTI.
We have recently shown that specific pathogens (specifically B. pertussis, influenza virus, RSV, adenovirus, parainfluenzavirus, bocavirus) are detected significantly more frequently from the NP of children with pneumonia than age-matched controls. In this study we detected all of these organisms, irrespective of TB-classification, suggesting that these pathogens may play a role in the exacerbation of symptoms in children with TB as well as accounting for the respiratory illness of a subset of the children without TB.
Whilst we detected multiple significant co-occurrences between different microbes in this study, these require more detailed assessment in a larger group of children. For example, the co-occurrence between M. catarrhalis, S. pneumoniae and H. influenzae, may reflect age-specific colonization patterns as previously reported [25, 26]. Other microbial co-occurrences, such as the association between S. aureus and P. jorovecii, were unexpected. We are currently conducting a larger, longitudinal study to better understand these co-occurences.
We were unable to identify significant associations between individual nasopharyngeal microbes and TB classification. Discriminant analysis identified that the presence of C. pneumoniae, hMPV, coronavirus O43, influenza C virus, rhinovirus and cytomegalovirus best discriminated children with definite TB. The significance of co-detection of these microbes in children with TB is unclear, and needs to be further assessed. One possibility is that the relative immune suppression or lung pathology associated with PTB may render the host susceptible to other respiratory infections, or alternatively, that intercurrent infection may be immunosuppressive, predisposing to an accelerated clinical course or likelihood of symptoms in children with PTB. Active TB is associated with suppression of cellular immune responses, which are critical for the control of intracellular infections such as many of those associated with definite TB in this study. However, in this study, individual microbes were each only detected in small numbers of children which limits our ability to draw firm conclusions in this regard.
A recent South African study has shown an increased risk of death in adults with TB-influenza A virus co-infection (adjusted relative risk ratio [aRRR] 6.1) compared to TB infection alone. In contrast, de Paus et al. did not find a correlation between the seroprevalence of influenza antibodies and the development of clinically active TB in an Indonesian cohort. They did however show an association between elevated antibody titres against influenza A and the clinical stage of TB lung disease suggesting recent re-infection with influenza precedes clinical presentation with PTB.
A limitation of this study is the lack of a control group of children without lower respiratory symptoms. We are therefore unable to infer whether the pathogens detected played a role in the development or exacerbation of symptoms in this cohort. Further limitations include sampling of the nasopharynx rather than the lower respiratory tract, limiting the ability to infer causality for lung co-pathogens. Klebsiella pneumoniae, Legionella spp and Salmonella targets were excluded from analysis due to problems with assay specificity for these targets.
Pertussis and respiratory viral pathogens were present in respiratory specimens in all age groups and all study months (Table 1, Fig. 1). Although the rate of viral infections in Cohort 1 is not known, the overall viral detection rates in Cohorts 2 and 3 were high, at 64.6 and 74.8 % respectively overall (p < 0.001, Table 1), and 66.2 and 78.6 % respectively among those <24 months (p < 0.001, Table 1). Detection rates of specific viruses also differed between Cohorts 2 and 3. In particular, the incidence of influenza (Flu A and Flu B) was higher in Cohort 2 (17.0 vs. 9.6 %, p < 0.001). By contrast, Cohort 3 had a higher incidence of RSV (25.3 vs. 14.1 %), PIV (6.7 vs. 2.7 %), or multiple viruses (11 vs. 5.5 %), all p < 0.001 (Table 1).
All 413 nucleic acid extracts were analyzed using the RespiFinder19® assay (Figure 2). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) (Figure 2). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen (Figure 2). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown).
Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) (Figure 2).
Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2). In addition, RespiFinder19® assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified (Figure 2).
In mixed infections, PIV (1 to 4) and RHV were the most frequent (75% [27/36] and 61.1% [22/36], respectively), followed by H1N1v (36.1% [13/36]), ADV (27.8% [10/36]) and RSV-B (5.6% [2/36]) (Figure 2). Co-detection or multi-detection were very frequent along with adenovirus infection (50% [10/20]), PIV infection (37.0% [27/73]) including mixed infections with several types and less frequently with rhinovirus infection (15.6% [22/141]). The frequency of viral co-infection was slightly higher in samples positive for H1N1v as compared to samples positive for other respiratory pathogens, but without significance (19.1% [13/68] vs. 10.8% [23/213]). RHV was, for instance, the more frequent co-pathogen in H1N1v positive patients (13.2% [9/68]). To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).
Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased (Figure 3). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50–60% down to less than 20% with a one to two weeks gap between SLS and TRS.
All 19 cases of suspected pertussis were confirmed by molecular analysis, of which for 2 B. pertussis strains have been isolated and analyzed for the evaluation of molecular variants. In particular, molecular analysis detected ptxA1, ptxP3 and prn2 allele variants (Table 4). Only 2 B. Pertussis strains were cultivated because 13 infants enrolled were already receiving specific antibiotic therapy when the specimen was collected or because technical problems arose during sample collection.
Our results confirm that respiratory infections were very common among French Hajj pilgrims, with more than 80% of them reporting at least one symptom during the 2014–2017 Hajj seasons. Our result is in line with the previous results obtained from cohort studies conducted among French pilgrims21 and from a large cohort study enrolling pilgrims from 13 different countries22. We also document the significant acquisition of almost all viral and bacterial pathogens included in our survey, following participation to the Hajj, as reported previously3,23.
We found that male gender was independently associated with a decreased risk for ILI and for the acquisition of coronaviruses and K. pneumoniae. We have no explanation for this unexpected observation. Older age (≥60 years) was associated with the acquisition of influenza viruses, HCoV and S. pneumoniae. Although older age was not correlated with the increase in the proportion of respiratory symptoms in our study, our results support the current French recommendations that indicate influenza vaccination for all pilgrims and IPD vaccination for pilgrims aged ≥60 years old (or with chronic conditions)24,25. Furthermore, we showed that influenza vaccination was significantly associated with a lower prevalence of ILI in our survey. Similarly, in a meta-analysis, Alfelali et al. have shown that, influenza vaccine decreases the prevalence of ILI26. We also demonstrate here that vaccination against IPD with a decrease in the acquisition of S. pneumoniae, but had no effect on respiratory symptoms. A protective effect of pneumococcal vaccination against S. pneumoniae post-Hajj carriage was observed in only one out of 5 studies so far9. However, all studies showing no effect of pneumococcal vaccination were conducted on small groups of pilgrims (ranging 55 to 107 individuals), while in the two studies showing a protective effect, the number of pilgrims was much higher (1178 and 468) which may indicate that small surveys lacked statistical power. Finally, as expected, patients suffering from chronic respiratory diseases were more likely to suffer from cough and ILI but also to acquire H. influenzae. Our results confirm the need for influenza and IPD vaccination among the identified pilgrim populations at risk of RTIs and acquisition of respiratory pathogens. Vaccination rates in our cohort were clearly sub-optimal: 26.7% of pilgrims were vaccinated against influenza and 30.5% against IPD among those with an indication.
With regard to non-pharmaceutical preventive measures, the use of masks, especially in crowded areas, frequent hand washing with water and soap or disinfectant, especially after coughing and sneezing, and the use of disposable handkerchiefs are recommended by the Saudi Ministry of Health to Hajj pilgrims27. In a meta-analysis including 13 surveys of Hajj pilgrims, significant protection of face masks was found against RITs, but the end points varied considerably12. We found that there was a higher prevalence of ILI and rhinovirus acquisition among pilgrims who reported wearing face masks. Similarly, a higher prevalence of cough was observed among pilgrims who reported using disposable handkerchiefs. It is likely that such results indicate the higher willingness of symptomatic pilgrims to wear a face mask and use disposable handkerchiefs, with the aim of avoiding spreading diseases. In addition, the use of disposable handkerchiefs appears to be correlated with a decrease in the acquisition of S. aureus carriage, which may reflect a better elimination of organisms by cleaning the nasopharynx. We found that increased hand hygiene was not associated with reduced respiratory pathogens acquisition or a lower prevalence of respiratory symptoms. In a recent review paper, it was shown that while hand hygiene using non-alcoholic products was generally well accepted by Hajj pilgrims, there was no conclusive evidence of its effectiveness, which is consistent with our results11.
Relationship between respiratory symptoms and carriage of respiratory pathogens at the Hajj are unclear. Because of the high frequency of respiratory symptoms, the distinction between infection and colonization is difficult to assess. Furthermore, asymptomatic carriage of potential pathogens is also observed among Hajj pilgrims28. Nevertheless, in the final model of multivariate analysis, the acquisition of S. aureus was associated with ILI and the acquisition of rhinovirus was associated with respiratory symptoms. Many studies showed that S. aureus and rhinovirus were among the predominant pathogens isolated from the Hajj pilgrims suffering RTIs9,11,12,21,23–32. Most cases of infections due to HRV are benign, self-limited cold-like illnesses. Nevertheless, HRV is also responsible for severe pneumonia in the elderly and immunocompromised patients, as well as exacerbations of chronic obstructive pulmonary disease and asthma33. HRV spreads mostly via direct contact or contact with a fomite, with inoculation to the eye or nose from fingertips34. The human-to-human transmission of rhinovirus among pilgrims may have been favored by the crowded conditions at pilgrim accommodations or during performing the Hajj rites.
S. aureus is also often part of the human microbiome in the anterior nares35. However, persistent S. aureus nasal carriage is associated with secondary staphylococcal respiratory infection, predisposing to invasive disease, especially in the case of detection of IAV36–38. To date, few studies addressed virus-bacteria carriage in relation with RTIs at the Hajj4,28. In our study, overall carrying virus-bacteria was associated with ILI. Dual H. influenzae-K. pneumoniae carriage was associated with twice the risk of coughing and H. influenzae-S. pneumoniae carriage with 5 times the risk for respiratory symptoms. Further works aiming at better understanding the role of overall carrying virus and bacteria in the pathogenesis of the RTIs at the Hajj are needed.
Our study had some limitations. The study was conducted among French pilgrims only and cannot be generalized to all pilgrims. Observance with individual preventive measures was self-reported and frequency of changing face mask and exact quantification of hand hygiene practice were not assessed. qPCR used to detect respiratory pathogens does not distinguish between dead and living micro-organisms. S. pneumoniae serotypes were not investigated. Moraxella catarrhalis that was recently shown to be relatively frequently acquired by Hajj pilgrims39 was not included in our study. Finally, we did not to be any long-term follow-up to determine what infections might have had delayed presentation after return from the pilgrimage. Nevertheless, our study included a large samples size of pilgrims spanning four Hajj seasons giving it a stronger statistical power. A number of risk factors have been identified to recognize pilgrims at increased risk of RTIs and of acquisition of most common respiratory pathogens encountered in this setting. Also, the study confirmed the effectiveness of vaccination against influenza in reducing ILI symptoms and that of vaccination against IPD in reducing acquisition of S. pneumoniae. Given the limitations of the current study, the use of face mask and a reinforced hand hygiene should still be recommended for Hajj pilgrims until large-scale controlled studies are conducted to truly assess the effectiveness of these measures in the context of Hajj. The use of disposable handkerchief is, de facto, highly frequent among ill pilgrims and at least allows decreasing the acquisition of S. aureus.
Median age at hospitalization was 2 months (Table 1). Terms were hospitalized at younger ages than preterms (median 2.0 m vs 3.0 m). Median duration of hospitalization was 5 and 6 days for terms and preterms, respectively. Overall, 46% of infants were vaccinated at admission, with statistically significant higher frequencies in preterms (62%) than in terms (44%; p = 0.01). Among vaccinated infants, the median interval between first vaccination and hospitalization was 35d in terms (mean 106d), compared with 37d in preterms (mean 105d).
Macroscopic pneumonia in the strict control group (non-infected, non-vaccinated) was minimal (0.08 ± 0.15), and macroscopic lesions were detected in only 2 of the 6 pigs inoculated with only B. bronchiseptica (Bb/NV/NCh), with a group average of 1.8% of the lung affected (Figure 2). There was not a significant increase in the percentage of gross pneumonia in the NV/Ch group when compared to the control group, though the percentage of pigs in each group presenting with lesions was different (100 vs. 25%, respectively). Also, there was not a significant difference between NV/Ch and LAIV/Ch groups (p > 0.05). The average percentage of lung affected by lesions for the NV/Ch group was 4.1 ± 2.9 compared to 2.8 ± 4.3 for the LAIV/Ch group. There was an increase in the percentage of lung affected in the Bb/NV/Ch and Bb/LAIV/Ch groups when compared to the strict control group (p < 0.05), but not between the Bb/NV/Ch and Bb/LAIV/Ch groups (p > 0.05).
Microscopic lesions were either not present or minimal (limited to mild interstitial thickening) in the strict control group (NV/NCh; Figure 3A), as well as in all but 2 of the pigs inoculated with B. bronchiseptica alone (Bb/NV/NCh). The two Bb/NV/NCh pigs with microscopic changes had lesions consistent with chronic B. bronchiseptica pneumonia characterized by moderate thickening of the alveolar septa with fibrin and collagen, type II pneumocyte hyperplasia, and alveolar spaces variably filled with macrophages (30) (Figure 3B). Pigs inoculated with IAV alone (NV/Ch) had mild lesions consistent with IAV infection characterized primarily by suppurative bronchitis and bronchiolitis with epithelial necrosis and peribronchiolar lymphocytic infiltration (31) (Figure 3C). The presence and severity of interstitial pneumonia was minimal to mild in the NV/Ch group. The IAV-associated lesions were diminished in the vaccinated group (LAIV/Ch) when compared to the non-vaccinated group (NV/Ch). In particular the suppurative bronchitis or bronchiolitis with epithelial necrosis was reduced; however, there was peribronchiolar lymphocyte infiltration and bronchus associated lymphoid tissue (BALT) hyperplasia in the majority of LAIV/Ch pigs (Figure 3D).
Pigs that were infected with B. bronchiseptica and subsequently challenged with IAV (Bb/NV/Ch) had microscopic lesions consistent with both IAV infection as well as acute and chronic B. bronchiseptica pneumonia (Figure 3E). Influenza lesions included suppurative bronchitis and bronchiolitis with epithelial necrosis and submucosal lymphohistiocytic inflammation, as well as peribronchiolar lymphocytic infiltration. However, the suppurative bronchitis and bronchiolitis tended to be more severe than that observed in pigs infected with IAV alone, and furthermore alveoli were variably filled with neutrophils and macrophages with areas of alveolar epithelial necrosis, hemorrhage, and type II pneumocyte hyperplasia, which is consistent with acute Bordetellosis. In addition, in sections from some Bb/NV/Ch pigs there were areas consistent with chronic Bordetellosis characterized by interstitial pneumonia consisting of alveolar septal thickening with mononuclear cells as well as fibrin and collagen (Figure 3E).
Finally, as noted in the LAIV/Ch group, vaccinated pigs that had been infected with Bordetella and challenged with IAV (Bb/LAIV/Ch) had diminished bronchial and bronchiolar epithelial necrosis. However, these pigs had lesions consistent with both acute and chronic Bordetella pneumonia, including acute lesions consisting of alveoli and bronchioles that were variably filled with neutrophils and/or macrophages and alveoli with areas of epithelial necrosis, hemorrhage, and type II pneumocyte hyperplasia (Figure 3F). Sections from some of the pigs in Bb/LAIV/Ch group also contained chronic lesions of interstitial pneumonia consisting of alveolar septal thickening with mononuclear cells as well as fibrin and collagen, which is consistent with chronic B. bronchiseptica infection.
Four children from Cohort 2 (respiratory viral testing ordered but not pertussis-specific testing) had pertussis detected by RM-PCR (based on data recovered by the manufacture post hoc), including one case previously diagnosed at another institution. The median age of these 4 subjects was 6.6 years (range 6 weeks to 14 years), and the median duration of respiratory symptoms prior to testing was 5 days (range 2–14 days). All four subjects had cough as a clinical finding but coughing paroxysms were not reported, and there was no indication of receipt of macrolides in the medical record.