This prospective study was carried out in the Union Territory of Chandigarh (located in north India with a population of 1.05 million residing in urban, rural and urban–slum areas, of whom 11.3% are children), over 24 consecutive months from 1 April 2011 to 31 March 2013. The study was coordinated from the Advanced Pediatrics Centre (APC) at PGIMER Chandigarh, a tertiary care centre with nearly 20 000 annual in–patient admissions and 100 000 out–patient visits.

Enrolment of children aged 1 month to 12 years, fulfilling the WHO IMCI case definition of CAP designed for children <5 years [6-8], was carried out through active and passive surveillance ( Figure 1 ). Tachypnea was defined as respiratory rate >60/min for infants <2 months; >50/min for infants 2–12 months; >40/min for children >12–60 months; and >30/min for children >60–144 months. Active surveillance was conducted in 30 anganwadi clusters, selected to represent the population of Chandigarh, where trained research team members visited households daily, inquiring for clinical symptoms of pneumonia. Passive surveillance was carried out by research staff stationed in the Out Patient and Emergency Departments of the APC, by evaluating clinical signs of CAP in children presenting to these Departments. If symptoms were reported and tachypnea confirmed, the child was presented to a Medical Officer for confirmation and inclusion. Children with duration of illness >7 days; those who had received antibiotics for >24 hours at presentation or those with previous hospitalization within the preceding 30 days, were excluded. Children with wheeze received a single dose of bronchodilator (Salbutamol 0.15mg/kg by nebulization), and those whose symptoms disappeared were excluded. All children received standard treatment including antibiotics, other medications as required and supportive care as per institution guidelines.

Figure 1.  The screening process for children enrolled through passive or active surveillance. Trained research team members identified children with cough and /or difficult breathing, combined with tachypnea. If the child fulfilled WHO IMCI definition of CAP; confirmation of the diagnosis by a medical officer was required. Children whose symptoms of CAP disappeared with a single dose of bronchodilator were excluded. After obtaining written parental consent to participate, a total of 2345 children were enrolled in the study and included in analysis.

A total of 36 676 children underwent active or passive surveillance for CAP. Figure 1 shows the step–wise process used to enrol children. A total of 2345 children were enrolled and comprised the cohort included in the analysis.

Table 1 presents the baseline characteristics of children enrolled through active or passive surveillance. Children <12 months dominated in both groups. Severe and very severe disease was detected more frequently in children enrolled through passive surveillance. A total of 1145/2345 children (48.8%) were enrolled during the cold season from 16 November to 15 February; while the remaining (51.2%) were enrolled during the longer warm season. Acute malnutrition, defined as weight–for–age z score less than 3, was observed in 1008/2345 children (42.9%). Similarly, absent or deficient breastfeeding (defined as duration of breastfeeding <6 months for infants older than six months, or less than infant’s age in those <6 months old) was more common in those enrolled through passive surveillance. These children were also more likely to be exposed to solid fuels as well as tobacco smoke in their homes. There were no major differences in gender, history of wheeze, previous history of infections, or family history of tuberculosis in children enrolled through active or passive surveillance.

Table 2 presents symptoms reported by parents, clinical findings and radiography. Almost all children presented with cough, fever and fast breathing with median duration of symptoms being similar in those enrolled through active or passive surveillance. Parents reported wheezing during the current episode in approximately one-third of the children. Symptoms/signs suggesting greater severity of pneumonia were more frequently identified in those enrolled through passive surveillance. A larger proportion of these children also had WHO Class I and Class II chest X–rays.

There were 108 (4.6%) deaths; of these 107 occurred among those enrolled through passive surveillance (mortality rate 9.2%) and one among those enrolled through active surveillance (0.1%). Based on disease severity, the mortality rate was 1.2% for pneumonia, 4.7% for severe pneumonia and 15.8% for very severe pneumonia. A comparison between fatal and non–fatal cases suggested that age <12 months, oxygen saturation <95% and radiographic finding of consolidation (WHO Class I) were associated with mortality.

Table 3 summarizes the microbiology findings from culture of different biological specimens. Blood culture results were available in 2285 children (97.4%) and only 49 (2.1%) were positive for pathogenic species. NPA culture results were available in 2323 children (99.1%) and of these, potentially pathogenic organisms were identified in 322 (13.7%). BAL culture (performed in children requiring endotracheal intubation), 0–5 days (median 2 days) after enrolment was positive in only 3/30 children. In addition, serology was positive for M. pneumoniae in 103 (4.3%) and C. pneumoniae in 26 (1.1%).

Figure 2 summarizes the data from microbiologic analysis of respiratory specimens. Of the 469 NPA samples selected for multiplex PCR, only 428 (91%) could be fully processed and 422 samples (98.6%) yielded organisms (panel A in Figure 2 ). A single bacterium or a single virus was found in only 42 (9.8%) and 28 (6.5%) children respectively. S. pneumoniae dominated (n = 35) followed by S. aureus (n = 5) and H. influenzae (n = 2). The single viruses identified were CMV (n = 13) and RSV (n = 9) followed by Rhinovirus (n = 2), and one each of Influenza, Parainfluenza, Enterovirus and hMPV. S. pneumoniae was the dominant organism identified in NPA culture as well. A comparison of the bacterial yield from NPA by PCR and culture is shown in panel B in Figure 2 .

Figure 2.  Microbiological findings in samples obtained from the sub–group (n = 428) of children with CAP. The number above each bar represents the number of children with a positive result. (A) Nasopharyngeal aspirate (NPA) Multiplex–PCR findings (bacteria and viruses) in the sub–group (n = 428). (B) Comparison of diagnostic yield of bacteria in NPA by PCR and culture indicates that PCR has a higher sensitivity; PCR (white bars), NPA (blue bars) and double positive samples (green bars). (C) NPA PCR findings in children with fatal outcome (n = 25). (D) BAL PCR findings in children who were intubated and underwent broncho–alveolar lavage (n = 30). Combinations of pathogens in (E) NPA samples (n = 428) and (F) BAL samples (n = 30). N – Nil, B – Bacteria, V – Virus, M – multiple organisms.

Among the 428 children with NPA PCR results, 25 died and PCR showed diverse organisms distributed in a pattern similar to the 428 children (panel C in Figure 2 ). Among intubated children undergoing bronchoscopy as part of clinical care (n = 30), only 2 samples were negative on PCR and the remainder showed organisms in a similar pattern to NPA PCR (panel D in Figure 2 ).

Since most NPA PCR samples yielded multiple pathogens, the data were analyzed with respect to etiology patterns rather than individual pathogens. These included combinations of two bacteria, two viruses, one bacterium plus one virus, or mixed i.e more than one bacteria and/or virus (panel E in Figure 2 ). The most common combination of pathogens in individual samples was S. pneumoniae and CMV (n = 100) followed by 2 bacteria or 2 viruses.

In BAL samples, the single pathogens identified were S. pneumoniae (n = 3), S. aureus (n = 2), H. influenzae (n = 2) and CMV (n = 3); the majority of samples (n = 18) showed multiple organisms (panel F in Figure 2 ) that were distributed in a pattern almost similar to NPA samples.

The complex microbial patterns on PCR were further analyzed with respect to disease severity (defined according to WHO criteria) but there were no apparent differences ( Figure 3 ).

Figure 3.  NPA Multiplex–PCR findings stratified by pneumonia severity as defined by WHO IMCI criteria in the sub cohort (n = 428). N – Nil, B – Bacteria, V – Virus, M – multiple organisms.

To our knowledge, this is one of the largest single–centre studies of CAP etiology in children from a resource–limited setting. Our data suggest that CAP is associated with a number of pathogens or combinations of viral and bacterial pathogens. Further, no single pathogen or combination could be related to disease severity. Our findings also confirm that infants <12 months old are particularly vulnerable in terms of disease severity and outcome.

As expected, S. pneumoniae was the predominant isolate in NPA by culture as well as PCR, although mere detection does not establish a causal role. We could not do serotyping due to resource constraints. It can be argued that the isolation rate by culture in our cohort is lower than expected [26,27], especially as PCR identified S. pneumoniae much more frequently. It is possible that clinical pneumonia due to other pathogens masks the presence of S. pneumoniae on routine culture. The major difficulty in attributing etiology to S. pneumoniae is its frequent presence in asymptomatic children also, although a similar argument could be raised for S. aureus too [28,29].

Detection of multiple pathogens in NPA by PCR makes it difficult to ascribe a causal role to any one organism. Our culture and PCR data also suggest that nasopharyngeal specimens may perhaps be inappropriate for confirming microbial etiology in CAP. Indeed, this is in concordance with several recent studies showing the presence of various viruses in asymptomatic children as well as those with upper respiratory tract symptoms [19,30,31]. It appears that even M. pneumoniae can be identified in the nasopharynx of healthy children [32].

Somewhat surprisingly, CMV was the most common virus in our cohort, where none had immune–suppressive therapy, known primary immune–deficiency and where the HIV prevalence during the study period is reported to be <0.25% in the community [33]. While CMV is well–recognized as a pathogen in these latter settings, its frequent occurrence in CAP raises the possibility that it may contribute to pneumonia pathogenesis singly or with other pathogens [34]. This novel finding also emphasizes that although PCR is highly sensitive, it can detect only those organisms that are looked for–a limitation that is being overcome by next generation sequencing. After CMV, RSV was most frequently identified as previously reported also [2] while Influenza A and B were less frequent. Unfortunately, even BAL samples in a limited number of children could not ascertain etiology as most children had multiple organisms. Further the time–lag between presentation and obtaining BAL samples in the majority of children raises the possibility that some of the organisms could represent secondary infection.

How to interpret the detection of multiple organisms in respiratory tract samples from a given child? It is possible that infection by one (potential) pathogen facilitates other pathogens, or that mild infection with one organism becomes more severe in the presence of additional organisms. This is well documented with Influenza infection [35,36] and suggested for other organisms also [2]. However, the pattern of PCR findings did not differ with disease severity which is in concordance with initial data from the PERCH project also [16]. In our cohort, a single organism (bacteria or virus) was identified by NPA PCR in only a minority of children. Further NPA data may be skewed on account of nasopharyngeal carriage. The limited BAL data suggests that S. pneumoniae, CMV, S. aureus and H. influenzae may be the dominant pathogens in severe cases of CAP. In children with fatal outcome, the same pathogens were identified along with RSV.

In the small number of positive blood cultures, S. aureus predominated, rather than S. pneumoniae or H. influenzae, expected in a vaccine–naïve pediatric population such as our cohort. Clinical experience suggests that S. aureus is frequently responsible for community acquired infections in India, although it has not previously been documented as the most frequent cause of bacteraemia in childhood pneumonia. In contrast, it is the most frequently recovered pathogen in parapneumonic effusions/empyema complicating pneumonia [37-39] and also commonly isolated in blood cultures from infants with bacteraemia [40]. Therefore it is reasonable to conclude that S. aureus may be an important pathogen in childhood pneumonia as well. However, international and national antibiotic treatment protocols for childhood CAP do not use specific antibiotics against this organism.

The data presented in this study raise some important points for further research on childhood CAP. First, the mere identification of organisms by highly sensitive techniques may not confirm etiology. Even comparing the yield among cases vs controls, as planned in the PERCH Project [41] can at best suggest an association, but not causation. In an individual child, even the presence of organisms commonly associated with pneumonia may be of limited value for predicting pneumonia severity/outcome. The presence of potential pathogens in the respiratory secretions of apparently healthy children also raises the possibility that microbes may not be solely responsible for disease. It is likely that combinations of host immune status and/or response to infection/inflammation tip the balance from asymptomatic colonization to disease in a given child.

Although this study had several methodological strengths limiting the risk of bias, it also had limitations. The disproportionately large number of severe and very severe pneumonia cases attest to greater enrolment through passive surveillance. Lack of controls is a limitation since it would have provided data on nasopharyngeal carriage of pathogens in asymptomatic/ healthy children in this population. Further, research team members could not be stationed in a given anganwadi throughout the study period, hence pneumonia incidence could not be calculated. We could perform only qualitative PCR, and that too in a small proportion (20%) of the cohort.

This large cohort study (CAPES) identified multiple pathogens in various biological samples of children with CAP. Our data suggest that it is difficult to attribute etiology to a single pathogen in the majority of cases as co–infection is common and independent of disease severity. Multiplex PCR proved to be highly sensitive in identifying potential pathogens from respiratory samples; but lacked specificity for establishing a causal relationship. A novel finding of CMV carriage/infection in nasopharyngeal secretions was observed. Our findings suggest that clinical practice guidelines for management of suspected bacterial pneumonia in developing countries should additionally consider anti–Staphylococcal therapy. Rational vaccination policies against S. pneumoniae, H. influenzae and (in the future) RSV could decrease overall burden of childhood pneumonia morbidity and mortality.