Introduction

Acute respiratory infections especially pneumonias are the second leading cause of mortality after diarrhea in children under five years of age [1], particularly in resource limited countries. According to WHO estimates, 156 million new episodes of pneumonia occur annually worldwide, of which 151 million episodes are in the developing world, with an estimated 11–20 million (7–13%) severe enough to require hospital admission [1, 2]. In the Eastern Mediterranean region of the World Health organization (WHO), incidence rates of clinical pneumonia are quite high (estimated 0.28 episodes per child-year): a close second to South-East Asia (0.36 episodes per child-year) and Africa (0.33 episodes per child-year) regions [2].

Pakistan has a population density of nearly 236 people/km2 and in absence of adequate health care services, factors such as low birth weight and childhood malnutrition (31%) promote increased incidence of diarrhea and respiratory diseases, with high mortality especially in the very young [3, 4]. Moreover, it is one of five countries with the highest burden of child deaths attributable to pneumonia and Acute Lower Respiratory Infections (ALRIs) along with India, Nigeria, the Democratic Republic of Congo (DRC) and Afghanistan [2]. With an estimated incidence rate for new cases of clinical pneumonia at 0.41 episodes/child year and predicted number around 9.8 million new cases annually [1, 5, 6], childhood ARIs/pneumonias continue to be a significant public health problem in Pakistan.

In the 1980’s, Ghafoor et al reported Pneumococcus and Respiratory Syncytial Virus (RSV) as the most common bacterial and viral pathogens respectively, among children with severe pneumonia in Islamabad and Rawalpindi [7, 8]. In 2010, Zaidi et al conducted a population based surveillance study in semi-urban areas of Sindh on incidence of pneumococcus-associated pneumonia among children, but viral causes were not explored [9]. Recently, Asad et al reported that respiratory viruses caused a significant percentage of WHO defined severe pneumonia in young hospitalized children in Karachi, Pakistan [16]. Data generated from studies in other developing countries shows that viral pathogens, primarily respiratory syncytial virus (RSV), human metapneumovirus (HMPV), influenza A and B viruses and adenoviruses are major contributors to upper respiratory tract infections. Currently limited information exists on the role of respiratory viruses in severe pneumonia or mortality in children below 2 years in high incidence countries such as Pakistan [10–15]. More so, inadequate data is available on the viral etiology of childhood pneumonias in northern regions of Pakistan, an area climatically and geographically distinct from the south (Karachi).

This study was conducted at two public sector tertiary care hospitals in Islamabad Pakistan to determine the viral etiology in children up to 2 years of age diagnosed with bronchiolitis and pneumonia, with or without radiological confirmation from March 2011-April 2102.

Materials & methods

Patients and definitions

The viral etiology and clinical features were evaluated in children from 6 weeks to two years of age, admitted to Pediatric Departments of two major public tertiary care hospitals in Islamabad; Federal Government Services Hospital (FGSH) and Pakistan Institute of Medical Sciences (PIMS) Islamabad, with acute lower respiratory infections between 1 March 2011 and 30 April 2012. Detailed epidemiological and demographic data was collected from each case using a pre-tested questionnaire form (Table 1). Samples from enrolled subjects were collected after informed and written consent from the children’s parents/guardians. The study design was approved by the Internal Review Board of National institute of Health, Islamabad, Pakistan.

Table 1 Individual risk factors and variables considered for samples collected in this study
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Lower respiratory tract infection (ALRI) was diagnosed using WHO defined criteria for severe pneumonia, i.e tachypnea (respiratory rate >60/min in children <2 months, 50/min in children 2–12 months and >40/min in children >12 months) and chest in drawing or any related critical sign, were used to identify children 6 weeks to 2 years old with at least one of the following: fever during the last 48 hours, coughing, runny nose, plus one of the following: wheezing, tachypnea, dyspnea, cyanosis, intercostal retractions, congestion, and/or crepitations on lung auscultation. Patients with URTI, history of longer than one week, parents refusal to participate, or complicated cases like pleural empyema or lung abscess were excluded from the study.

The laboratory results such as serum Haemoglobin levels, total leukocyte counts with differential and arterial blood gases (if done) levels were also evaluated in most patients with ALRI. Chest radiographs of most cases were evaluated with the attending clinician. Therapeutic management records using antibiotic therapy, inhaled steroid and/or bronchodilators were reviewed. Duration of hospitalization and any complications developed (need for assisted ventilation etc.) and disease outcome were recorded.

Specimen collection for viral analysis and processing

An informed consent was obtained from each parent prior to sampling. Nasopharyngeal or oropharyngeal swabs were collected using flocked swabs (Copan®), placed in viral transport medium and transported maintaining the cold chain to the virology laboratory at National Institute of Health Islamabad on the same day. Swab specimens were stored at 4 °C and analyzed within 72 hours of collection.

RNA was extracted directly from 140 μl of sample supernatants with an RNeasy Viral mini kit (Qiagen, Valencia CA, USA), according to the manufacturer’s instructions. The RNA was eluted in 60 μl DNase and RNase-free water. 5 μl of the extracted RNA was used as template in 25 μl real time PCR mix. The reaction was performed on ABI-7500 using a panel of oligonucleotide primers and dual labeled hydrolysis (Taqman®) probes according to the CDC protocol for Non-Influenza respiratory viruses (kindly shared by Drs. Erdmann and Peret from CDC). The assay tested each sample separately for RSV, adenovirus and Human Metapneumovirus. Human RNase-P gene served as an internal positive control for human nucleic acid. No template/negative controls (NTC) and positive template controls (PTC) for all primer/probe sets were included in each run Real Time Reverse transcriptase polymerase chain reaction was performed for Influenza A/B and further subtyping of influenza A using the CDC protocols [17]. Identified Influenza viruses were cultured on Madin- Darby Canine Kidney (MDCK) and RSV on Hep-2 cell line.

Statistics

Statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS) version 17.0. Chi-square and Fisher’s exact tests were utilized for comparison of categorical variables. Statistical significance was defined as p < 0.05.

Results

A total of 155 children under 2 years admitted with a clinical diagnosis of bronchiolitis or pneumonia to pediatric units of two major tertiary care hospitals of Islamabad were enrolled in the study during March 2011 to April 2012.

Patient demographics

Majority of the patients (62%; n = 96) were males. The overall mean age was 7.83 ± 5.25 months (range: 1-54 months, median: 6 months). The age group distribution of the subjects was as follow: were 1-2 months; 21.3% (n = 33), 2.1-6 months; 29.7% (n = 46), 6.1-12 months; 33.5% (n = 52) and 12.1-24 months; 15.5% (n = 24) [Figure 1]. The average weight of the study group was 6.1 ± 2.25 kg (range: 2-10 kg). The highest number of positive cases correspondingly fell in the 4-6 Kg range. History of underlying morbidities among the patients with a detected viral agent included a congenital anomaly; 6 (5%), cardiac defects; 2 (1%), underlying respiratory disease (asthma, previous H/O pneumonia or TB); 7 (6%), CNS related (H/o epilepsy or fits) in 2 (1%) patients and a single case of congenital renal disease. Highest number (85.2%) of virus positive cases was admitted between November and January (Figure 2).

Fig. 1
figure 1

Detection of Respiratory Pathogens by age groups. Detection of Individual viruses as a proportion of all viruses detected in patients of all age group

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Fig. 2
figure 2

Seasonality of virus detection, March 2011-April 2012; Month wise detection of all respiratory viruses investigated under this study during March 2011 to April 2012

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Virus detected

One or more viral pathogens were detected in 117 patients (75%) of 155 cases with detection frequencies for RSV A; 44% (n = 69), RSV B; 23% (n = 35), Influenza A 24.5% (n = 38), Influenza B: 7% (n = 11) Adenovirus; 8.4% (n = 13) and HmPV: 5.2% (n = 8). A single viral pathogen was detected in 43% (n = 67) patients, 2 pathogens in 29% (n = 45) and upto 3 agents in 3.2% (n = 5) cases. Viral detection was most common (37%) in cases admitted to the hospital during December and January (Fig. 2).

Virus detected in clinical profile

A viral agent was detected in 45% (n = 67/148) and 55% (n = 81/148) of patients admitted with clinical diagnosis of bronchiolitis and pneumonia respectively. When Bronchiolitis Vs pneumonia frequency was compared for each virus, the distribution for RSV and Influenza B was almost equivalent (bronchiolitis: pneumonia; 45:46.4), while pneumonia percentage was higher (60.5%) among the Influenza A positive cases. Over all, the most common presenting symptoms were cough (95%; n = 147), apnoea (84%, n = 131), fever (78%; n = 121), wheeze (64.5%; n = 100), nasal congestion (55%; n = 86), poor feeding/refusal to feed (63.2%; n = 98), rhinorrhea (48%; n = 21/44) and Cyanosis 29%; 35/121. (Tables 2 & 3).

Table 2 Summary of data and analysis of predisposing practices/factors for viral pneumonia in the hospitalized children
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Table 3 Comparison of clinical features in hospitalized children with ALRI for various respiratory viruses
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Among the RSV positive cases, 2-6 months age group had highest detection rate for RSV A (30%, n = 21/69) and RSV B (20%, n = 14/69). The most frequent clinical features associated with RSV infection were cough (64%), fever (54%), wheeze (43%), apneoa (55%) and poor feeding (43%). Statistically significant difference was observed between RSV-positive and negative cases for nutritional status- weight for age (p = 0.001), cigarette/wood smoke exposure (p = 0.001) and concomitant clinical findings such as rhinitis (p = 0.023), ear infection (p = 0.040), conjunctivitis (p = 0.037), crackles (p = 0.005) and stridor (p = 0.009) (Table 2 & 3).

Majority of Patients infected with Influenza A were in 2.1 - 6 months age (61%, 23/38); and 6.1 - 12 months age group (55%, n = 6/11) for Influenza B. Overall, the most frequent symptoms in influenza positive cases were cough (95%), fever (79%) nasal congestion (53%), and wheeze (59%). Overall, statistically significant difference was observed between the Influenza positive and negative cases for breast feeding vs bottle fed (p = 0.033), influenza vaccination status (p = 0.038) and concomitant clinical management parameters such as abnormal ABG values (p = 0.001) and requirement of mechanical ventilation (p = 0.010) (Table 2 & 3).

HAdV was detected in thirteen patients; a double infection in six (5 with RSV A, 01 with Inf B), triple infections in 6 cases (05 Inf A + RSVB + AdV and 01 Inf A + RSV A + AdV) and as sole viral agent in only one patient. Six patients had bronchiolitis (all co-infections) and five had severe pneumonia (one single infection and four co-infections) while clinical diagnosis was unavailable for one patient. Clinical findings such as crackles (p = 0.033) and chest indrawing (p = 0.010) were found to be statistically significant between the HAdV positive and negative cases (Table 3).

Patients with HMPV infections had a myriad of symptoms, including fever, cough, rhinorrhea, wheezing, and sore throat, none of which showed any significant association. Out of eight, four were patients with single infections, two co-infections with RSA and RSV B respectively, and remaining two were triple infections with Inf B + RSVA and Inf A +RSVA respectively.

One fifty five specimens were tested for rhino virus by Real Time RT-PCR; 19 (12%) were found positive in which previous admission (p = 0.037), poor feeding (p = 0.043), stridor (p = 0.002) and ABG’s (p = 0.040) were found statistically significant between rhino positive and negative cases (Table 1, 2 & 3). The overall median age of cases was 6 and IQR was 6.50 (in months).

Laboratory and radiographic findings

On analysis of total White blood cell/leukocyte count (TLC) data from the patients, TLC was raised above normal values in 51% patients, normal for 39% cases and not available for the remaining 10%. Differential counts for raised Neutrophil (N) and lymphocyte (L) level showed; Raised L and low Ns- 22%, Raised Ns and low Ls- 32%, Raised Ls only-26%, Raised Ns only-8%. The neutrophil-lymphocyte ratios were mixed and did not provide any supplementary evidence that could be helpful in identification of etiology. Hemoglobin levels were below normal range in 110 out of 155 patients (71%).

Statistically significant difference was observed for RSV negative and positive patients who had deranged/abnormal ABG values (p = 0.031) and a similar trend was seen for ABG values, among the Influenza (p = 0.001) and HmPV (p = 0.002) positive cases(Table 4). Radiographic findings were available for 77% (n = 120/155) cases with evidence of; Lung Infiltrates -38.1% (n = 59), opacities in lung fields-23.2% (n = 36), consolidation-12.3% (n = 19), and normal-3.9% (n = 6). The radiographic findings showed a higher percentage of lung infiltrates among RSV A positive cases (40%) followed by Influenza A (32%).

Table 4 Clinical diagnosis, laboratory results, and treatment of hospitalized children with ALRI for various respiratory viral pathogens
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Treatment/management

Most Patients had a successful clinical outcome on combination therapy with bronchodilators, inhaled steroids and antibiotics. Majority of the patients were treated with antibiotics due to very young age, bacterial co-infection or following transfer to an intensive care unit (ICU) due to worsening clinical condition. Inhaled bronchodilator therapy was given in 91% (141/155) of the cases along with use of steroids; 71% (110/155), Oxygen inhalation; 43% (67/155) and assisted mechanical ventilation; 34% (52/155). Only for influenza positive cases, requirement of mechanical ventilation found to be statistically significant (p = 0.010). Treatment protocols for patients with Influenza virus or RSV associated ALRI did not include antivirals Oseltamivir® or Palivizumab® monoclonal antibody respectively. Only one patient with severe other morbidities but no detected viral etiology died in the ICU.

Discussion

The public health problem presented by high incidence of pneumonia and lower respiratory tract infections in young Pakistani children is complicated by dearth of recent and nation-wide epidemiological data on its incidence and etiology. The objective of the study was to define the viral etiology among children hospitalized with ALRI (pneumonia + bronchitis) and correlate with clinical severity and case management. The clinical spectrum of many common childhood illnesses overlaps significantly with pneumonia including malaria, bacterial sepsis, and severe anemia; and differentiating between these conditions on clinical criteria alone can be quite challenging [33–36]. The dilemma that a large percentage of pneumonias rely on rapid breathing as a main clinical sign for diagnosis has led to increasingly prevalent practice of injudicious antibiotic use in a large percentage of ALRI cases without laboratory evidence compounding increased antimicrobial resistance [18].

The data presented here is the first detailed description of clinical features associated with viral pathogens in children hospitalized with ALRI in Pakistan using real time RT-PCR assay as a diagnostic tool [19]. This study focused on hospitalized cases with a clinical diagnosis of bronchiolitis (45.4%) and pneumonia (54.6%) on admission in which a viral agent was eventually detected. We found that 75.4% of patients between 1-24 months age were positive for one or more viral pathogen including RSVA/B, Influenza A/B, HmPV or HAdV. The detection rates for viruses are higher when molecular diagnostics are employed, with 30–50% of the severe pneumonia cases attributable to a viral agent [14, 19, 32]. Consistent with our study, previously published hospital-based studies using molecular diagnostic techniques in Bangladesh, Turkey, Iran and Mozambique reported high detection rates for viral pathogens ranging between 36.7-77% [13, 20–22, 37, 38]. Even though molecular diagnostic tests have greatly improved our understanding of the role of viruses in pneumonia with respiratory syncytial virus, rhinovirus, human metapneumovirus, human bocavirus, and parainfluenza viruses being some of the most frequently identified agents in both developed and developing countries. However, generally unavailability or inadequacy of appropriate diagnostic facilities is a significant challenge in defining the viral etiology of severe pneumonia in developing countries which leaves many ALRIs undiagnosed and under-estimation of the viral pneumonia incidence rates [39].

Epidemics of respiratory viruses have been largely observed during winter and early spring seasons [1, 15, 28, 32–34] and a majority (85.2%) of positive cases were admitted in November, December and January, suggesting that routine poly viral workup in these patients might be cost-effective and would thwart the overuse of unnecessary antibiotic therapy at least during this period. There is need for availability of at least limited testing for principal viral and bacterial pathogens, or clear cut recommendations for antibiotic use in young children.

We concluded that the burden of RSV infections was significantly high in Pakistani children under 2 years of age particularly among children less than six months of age (67%, n = 104). This finding is supported by results from other countries which have demonstrated a high prevalence rate for this virus in early childhood pneumonia [23–25]. Notably, while a community based cohort study conducted in rural Bangladesh on children aged 0–24 months, detected RSV in up to (81%) cases with pneumonia[13], other researchers have reported somewhat lower prevalence rates of 25-36% for RSV [24, 26–29, 48]. The Bangladesh study however, included cases of bronchiolitis as in our study protocol, which might have been responsible for overall higher RSV detection rates. We found a predominance of RSV Group A infection amongst the patients, which has been reported frequently from other regions [30, 31] a trend that may vary year after year [32]. Since major RSV subtypes may vary from one season to the next and our data is based on samples collected for one year only, contiguous yearly data will be useful to describe any circulation trends and to estimate the RSV burden in order to consider potential public health benefit of RSV vaccination or prophylaxis for vulnerable population groups.

Important risk factors such as breast feeding practices, smoke exposure, history of prior vaccination or lack thereof and concomitant infections such as conjunctivitis and otitis were significant factors for RSV infections. The role of underlying risk factors towards increased incidence of childhood pneumonia has been extensively reported; including indoor air pollution [40, 41], malnutrition [42, 43], absence of breastfeeding, low maternal education, impact of low socioeconomic status, poor access to healthcare, and concomitant illnesses [44]. Efforts to improve maternal education by including materials on awareness of risk factors in pneumonia awareness campaigns could potentially help in reducing exposure in young children. A high percentage of RSV positive cases (approx. 58%) had radiographic findings suggestive of pneumonia including lung infiltrates (33%) as confirmed by previous data [13]. While the possibility of secondary bacterial infection cannot be ruled out, these findings reinforce the association of radiological evidence with viral pneumonias. We deduce that radiographic evidence and determination of O2 saturation/hypoxia levels by pulse oximetry can be optimal diagnostic methods for supporting diagnosis of pneumonia [45–47].

The incidence of influenza-associated pneumonia is important as Influenza is the only viral illness against which both vaccination and treatment are available [49–51]. Infection with influenza viruses predisposes children to secondary bacterial infections such as pneumococcal and staphylococcal pneumonia resulting in severe illness /adverse outcomes [53–55]. During this study, influenza A was detected at a lower rate as compared to RSV and in many cases there were confections by both viruses. The cumulative frequency of influenza A & B subtypes (31.6%) was comparable to similar studies from developing countries with detection rates ranging from 28-36% reported for pneumonia in younger children [52, 56] while some research groups have reported influenza infection rates as low as 10% [57]. The fact that most of the patients included in the study were unvaccinated for influenza (Influenza vaccination is not mandatory as part of Expanded Immunization program) and maternal vaccination is also not routinely performed, the high percentage of influenza A or B positive cases is hardly unexpected as most of the patients were below 02 years in age and therefore immunologically naive. We recommend that maternal vaccination may be helpful in very young children since the highest positivity rates were found in this age group. Despite very high incidence of pneumonia associated deaths in Pakistan, vaccination against none of the viruses focused in this study is routinely done in Pakistan. Out of the four viruses, no vaccine is available against metapneumovirus, RSV and adenovirus. Although the vaccine against influenza virus is available, the vaccination rate is recorded negligible in merely <1% population done on voluntarily basis. The authors therefore emphasize on including vaccination against influenza as a part of routine national immunization program under Expanded Program on Immunization, Pakistan.

Adenovirus shedding may continue for months in absence of overt infection which can complicate the diagnostic picture. Furthermore, subclinical infection with adenovirus may increase the susceptibility to bacterial infections predisposing to secondary bacterial pneumonia [48]. In the current study, HAdV was principally detected as a co-infecting pathogen in twelve out of thirteen patients with severe pneumonia diagnosed in over 50%. Whether HAdV was the primary pathogen responsible, or the co-infection led to tipping of balance towards severe disease merit studies on carriage rates and their association with productive/clinical disease. HMPV was found in only 5.2% of children hospitalized with ALRI in our study. In contrast, Asad et al found a much higher incidence of 14.2 % for hMPV during 2010-11 [16]. This is comparable to findings of prospective surveillance in hospitalized children from the United States and Israel, which reported HMPV detection rates of 6%, 13% and 15% respectively [58–60].

We reported viral co-infections in 32% of the cases with two (29%) or three pathogens (3%) which is comparable with previously published data from Nigeria [61] China [49–51] and Spain [62]. The detection of multiple potential pathogens in a single patient can also present problems with assigning causality to a single pathogen, whether all detected viruses had a synergistic role in pneumonia pathogenesis or were these actually co-infections to begin with? This is an expected yet bothersome downside to the use of molecular testing methods and multiplex PCR assays in diagnostics with variable sensitivity and specificity for various pathogens. It is a known fact that most viruses that cause pneumonia are more commonly associated with upper respiratory tract infections, and viral shedding may occur for a long period of time after symptoms have disappeared [63–70]. This raises the question whether use of lower respiratory specimens such as tracheal washes or Broncho-alveolar Lavage fluid (BAL), albeit technically challenging, could be more appropriate for definitive diagnosis and reviewing the specimens of choice for ALRI might be required.

There are certain limitations to this study. The surveillance was conducted for a little over 12 months and the seasonal pattern of respiratory viruses can vary from year to year. Multiple year surveillance data is needed to reliably establish seasonality of different respiratory viruses. In a resource poor setting it might not be possible to confirm each incident of clinical pneumonia by radiographic investigation. Even though we also did not evaluate for bacterial etiologies since the objective of the study was to identity the viral pathogens associated with ALRI in children.

Conclusions

To address UN Millennium Development Goal 4 on reducing childhood mortality, there is need for better and reliable data on the rates and causes of neonatal and childhood mortality. Respiratory viruses should be the focus of additional efforts to decrease pneumonia-associated morbidity and mortality in developing countries like Pakistan. Defining the burden of these viruses using reliable and sensitive diagnostic tests is an important first step and efforts should be directed towards introduction of at least a minimum testing panel incorporating the important respiratory viruses. Introduction of cost effective diagnostics for viral etiology of pneumonia would be a most valuable investment towards formulation of objective antibiotic treatment guidelines for ALRI. The current and future similar studies will contribute to better understanding of the impact of viruses in respiratory infections in infancy emphasizing the need for precise diagnosis particularly for the new emerging respiratory pathogens.