Original Article
Respiratory Syncitial Virus in Children with Acute Respiratory Infections R. Hemalatha, G. Krishna Swetha, M. Seshacharyulu1 and K.V. Radhakrishna1 Departments of Microbiology and 1Clinical Division, National Institute of Nutrition, Jamia-Osmania, Hyderabad, India
ABSTRACT Objective. To study the nutritional status of children with Respiratory Syncitial virus infection. Methods. One hundred and twenty six children with acute respiratory infection, between the age of 4-24 months, were investigated for RSV infection with bronchiolitis, pneumonia and upper respiratory tract infection. Nasopharyngeal aspirates were collected and cytokine responses were determined by ELISA. Upper respiratory tract infections were detected in 16.66%, bronchiolitis in 30.15% and Pneumonia in 53.17% children. Results. Of the 126 patients, 46.66% children were positive for RSV while 58.33% were negative for RSV. Children with bronchiolitis were more commonly positive for RSV compared to URTI and pneumonia. RSV was almost equally distributed among boys (42.5%) and girls (48.7%). More children were RSV positive when the mean age lesser (8.4 mo) was compared to RSV negative (9.93 mo). Well nourished children and children with normal birth weight had more RSV positives, though not statistically significant. In a sub sample analysis of cytokines done (n=25), Interleukin-2 and Interleukin-8 levels were higher in the RSV positive children and these levels declined after 5 days of illness. Conclusions. RSV is more commonly associated with bronchiolitis in younger infants with normal birth weight or more weight for age (WFA). Proinflammatory cytokine IL-8 was secreted at high concentrations in the nasopharyngeal aspirate in all the children. [Indian J Pediatr 2010; 77 (7) : 755-758] E-mail:
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Key words : Respiratory syncitial virus (RSV); Bronchiolitis; Weight for age; Well nourished children; IL-8
RSV is the most common virus isolated in respiratory infections1 and is frequently detected in hospitalized children.2 It is the main cause of bronchiolitis worldwide and causes up to 70-80% lower respiratory infections during monsoon and winter.3 In India, studies based on the isolation and sero survey have demonstrated RSV to be the main virus responsible for lower respiratory tract infections in children below 5 yrs of age.4 The severity of RSV infection in young children varies from a non clinical or mild respiratory infection to severe lower respiratory tract infection that may lead to hospitalization and occasionally death. Though, the mechanisms underlying the highly variable disease course in children is still poorly understood, it is believed that children with severe RSV disease suffer from enhanced inflammatory lesions
Correspondence and Reprint requests : Dr. R. Hemalatha, MD, Deputy Director, Head, Department of Microbiology, National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania (PO), Hyderabad- 50000 7, Andhra Pradesh, India. [DOI-10.1007/s12098-010-0108-z] [Received July 06, 2009; Accepted April 19, 2010]
Indian Journal of Pediatrics, Volume 77—July, 2010
rather than from virus induced cytopathy. 5 Children who experience RSV infection in early in life, run a high risk of subsequent asthma and recurrent wheezing. Moreover, the sera and nasal secretions of the RSV infected infants show a marked increase in the levels of Th-2 cytokines and chemokines.6 In vitro studies have shown that epithelial cells or macrophages infected with RSV secrete high concentration of IL-87. Studies on infants infected with RSV also show elevated levels of IL-8 in serum and nasopharyngeal aspirate and to the large number of neutrophils seen in the lavage fluid of these infants.8 It is now recognized that neutrophils play an important role in viral respiratory tract infections, but importance of their role in eliminating viral infection is still unclear and indeed they may have little or no effect in limiting viral replication. However, there is evidence that they play an important role in the causation of symptoms during respiratory viral infections. 8 Several studies have suggested that IL-2 is an appropriate means of determining inflammatory response in patients with severe acute viral bronchiolitis, in the acute phase of the disease.9 755
R. Hemalatha et al The mutually adverse interactions between malnutrition and infection are complex and operate through various mechanisms. The immune response, especially cell mediated immunity is known to be poor in undernourished children. There are, however, very few studies to show the relationship between RSV infection, nutritional status of the children and local cytokine response which plays a key role in the outcome of the disease. The present study was undertaken to investigate the nutritional status of RSV infected children having respiratory infections, with special attention to body weight, age, hemoglobin, vitamin A and local IL-2 and IL-8 from nasopharyngeal aspirate.
High Performance Liquid chromatography (HPLC). Anthropometric measurements were taken to assess their weight for age, using Gomez Classification. Nasopharyngeal Aspirate (NPA) was collected aseptically by passing size 5 feeding tube into the nasopharynx and applying gentle suction with a syringe. Secretions were rinsed into collecting vials with 1ml of phosphate buffer. After centrifugation of NPA to precipitate cells, the supernatant was frozen at -70 0 until analyzed for cytokines and serum samples by ELISA (Diaclone Research). Total protein from NPA was determined by Modified Lowry’s method. Statistical analysis of the data was done using SPSS software.
MATERIALS AND METHODS
RESULTS
The present cohort study was carried out at a tertiary care centre, Niloufer Hospital, Hospital for Women and Child Health, Hyderabad from the months of August through March 2007-2008. Inclusion criteria were: All children with respiratory infection who had visited the out patient(OP) department during the study period, diagnosis of Pneumonia, bronchiolitis or Upper respiratory infection(URI) with history of illness not more than five days. Exclusion criteria were: Children with congenital heart disease, chronic lung disease, family history of asthma, and other chronic disorders. This study was approved by the Institutional Review Board (IRB) and written informed consent was taken.
Over a period of eight months, a total of 126 children with acute respiratory infection were recruited. Of the 126 children recruited, 21 had URTI, 38 had bronchiolitis and 67 had pneumonia. A total of 56 children were positive for RSV while, 70 were negative. Distribution of clinical cases in the age groups <1 yr and ≥1 yr showed a significantly higher proportion of younger infants to be infected with RSV. The mean age of children with RSV was 8.4 months and 9.4 months for RSV negative children. Of the 126 cases, 22 children were treated as outpatients, and 104 were hospitalized. Of these 104, 99 children recovered and five children (3 RSV + and 2 RSV -) had fatal outcomes and died in the hospital (Table 1).
Acute respiratory tract infection was defined as the presence of cough with or without fever for less than 2 wks. Bronchiolitis was diagnosed in those who presented with the prodromal symptoms of rhinorrhea, cough, low grade fever, followed by paroxysmal wheezy cough, dyspnea, with signs of air hunger, chest retractions, wheezing on physical examination and hyperinflation of lungs with patchy atelectasis on chest radiograph. Pneumonia was diagnosed in those who presented with history of poor feeding, respiratory distress with minimal wheeze, cough with chest retractions, shaking chills accompanied with fever, clinical findings of decreased breath sounds, consolidation or scattered rales heard all over the chest over the affected areas, and features suggestive of consolidation or patchy or interstitial infiltration on chest radiography.
Children with bronchiolitis (60.5%) were more commonly positive for RSV compared to URTI (19%) and pneumonia
For all the cases, clinical history and physical examination was done and recorded. Weight was measured to the nearest 100 g with an electronic scale (Secca). Infants were weighed with the help of an infantometer. Low birth weight was defined as those infants who were weighing ≤ 2499 g at birth, irrespective of gestational age. From all cases, 2 ml blood was collected in a plain vial for measuring Hemoglobin (Hb) status and Vitamin A levels. Vitamin A levels were determined by 756
TABLE 1. RSV Prevalence and Its Association with Various Parameters RSV +ve(56) Respiratory infection(n) URTI(21) Bronchiolitis(38) Pneumonia(67) Outcome(n) Recovered(99) Death(5) OP(22) Age (n) <1 yr(89) >1 yr(37) Sex(n) Males(87) Females(39) Wt/Age (n) <75% (71) >75 %(55) Birth Weight (n) Low(26) Normal(94)
RSV–ve (70)
n (%) 4 (19.0) 23 (60.5) 29 (43.3)
n (%) 17 (81.0) 15 (39.5) 38 (56.7)
48 (48.5) 3 (60) 5 (22)
51 (51.5) 2 (40) 17 (77.2)
47 (52.8) 9 (24.3)
42 (47.2) 28 (75.7)
37 (42.5) 19 (48.7)
50 (57.5) 20 (51.3)
27 (38) 29 (52.7)
44 (62) 26 (47.3)
9 (34.6) 47 (50.0)
17 (65.4) 47 (50.0)
n followed by percentage in parenthesis
Indian Journal of Pediatrics, Volume 77—July, 2010
Respiratory Syncitial Virus in Children with Acute Respiratory Infections (43.3%). RSV positive cases were almost equally distributed among boys (42.5%) and girls (48.7%), (P=0.279). A higher proportion of younger children and well nourished children were positive for RSV (Table 1). The mean WFA and the serum Vitamin A levels were higher in the RSV infected children, while the hemoglobin status and height for age was comparable.
5000 a
IL8 1st day Il8 5th d ay
a
4000 a
3000 b
300
2000 a
250
a
a
Il2 1st day Il2 5th day
IL-2 in pg/gm protein
1000 200 b
0
150
pneu mo nia
b ro nchio litis
clinical diagnosis 100
Fig. 2. IL-8 levels in children with pneumonia and bronchiolitis on days 1 and 5. Means with different superscript are significantly different with P<0.05
50
0 pneumonia
bronchiolitis
DISCUSSION
clinical diagnosis
Fig. 1. IL-2 levels in children with pneumonia and bronchiolitis on days 1 and 5.
In a sub sample of 18 RSV positive and 7 RSV negative cases, nasopharyngeal IL-2 and IL-8 was done. The total concentration of IL-2 and IL-8 ranged from 45.4 to 722 pg/g total protein and 597 to 8127 pg/g total protein, respectively, with IL-2 being detectable in less than 50% of the cases( 12 out of 25). However, after 5 days, IL-2 was detectable in nearly all cases (22 out of 25 cases), but the total concentration decreased from the initial level. The mean concentration of IL-2 and IL-8 were higher in the RSV positive cases though not statistically significant (Table 2). Categorizing the cases based on their clinical outcome, it was found that on day 1, bronchiolitis and pneumonia cases had high IL-2 and IL-8 levels, however, children with bronchiolitis had relatively lower concentrations of IL-2 compared to children with pneumonia (Fig. 1). On day 5, children with pneumonia maintained same concentrations of IL-2 and IL-8 while children with bronchiolitis secreted lower IL-8 levels (Fig 2). Children with pneumonia, positive for RSV had significantly higher IL-8 concentrations compared to those without RSV infection. TABLE 2. RSV Infection and IL2, IL8 Response in Children with ARI RSV +ve Vit A(µg/dl) Hb (g/dl) IL-2 day 1 day 5 IL-8 day 1 day 5
19.95 11.01 285 213 3425.2 2670.9
± 1.513 ± 0.298 ± 64.76 ± 44.4 ± 436.5 ± 384.8
RSV -ve 16.42 ± 1.769 10.78 ± 0.237 235.3 ± 29.18 157.7 ± 33.21 2176.7 ± 666.84 1092.6 ± 180.5
IL-2 and IL-8: pg/gm of total protein. The values are Mean ± SE
Indian Journal of Pediatrics, Volume 77—July, 2010
In the present study, nutritional status of children with RSV infection, and the nasopharyngeal cytokine levels in them were studied. In a number of studies on hospitalized children with bronchiolitis or pneumonia, RSV has been found to be associated with a higher proportion of bronchiolitis compared to pneumonia.10 Similarly in the present study, RSV was more commonly associated with bronchiolitis than pneumonia or upper respiratory infections (URTI). Earlier studies showed that RSV prevalence was found to be more in males than females11 and male gender was considered an independent risk factor for the development of severe RSV infection. In contrast, the authors observed an equal distribution of RSV in boys and girls though association with younger age was similar to that reported elsewhere. 12 Though malnutrition has been found to be a risk factor for acute LRI, 13 animal studies on RSV infection and nutrition showed conflicting results.14 In the present study, good nutritional status (WFA) and higher Vitamin A levels were associated with RSV infection. A similar observation was made by Clarios et al who reported good nutrition, obesity and over nutrition as risk factors for RSV infection.11 The role of inflammatory mediators in the pathogenesis of RSV disease though not well-understood, has been studied by various people. In vitro studies have shown that epithelial cells or macrophages infected with RSV secrete high concentration of IL-8.7,15,16 Similar to the present study, other studies on infants infected with RSV also showed elevated levels of IL-8 in serum and nasopharyngeal aspirate that has been suggested in contributing to large number of neutrophils recruited in the lavage fluid of these infants. 8 That neutrophils 757
R. Hemalatha et al recruited by local IL-8 secretion might contribute to the symptoms during viral infection has been demonstrated by correlating neutrophil myeloperoxidase product with severity of upper respiratory tract symptoms.17 However, while some investigators suggested IL-8 polymorphism in the pathogenesis of hypersensivity reaction, others suggested sensitization of airway epithelium to environmental lipopolysaccharide exposure by altered IL8 expression in RSV infection.18, 19 Thus, even in mild to moderately nourished children, the airway inflammation that results from the viral infection is associated with a marked increase in IL-8 in the infected sites of the lung, which might contribute to increased local leukocyte response and hypersensitivity reactions. Under nutrition does not seem to be a risk factor for RSV infection, however, it does not seem to decrease the chances of these children developing sequelae at a later date. Acknowledgements We are grateful for the skillful assistance rendered by the nursing staff Santoshamma and Rajakumari in sample collection at Niloufer hospital. Contributions: R H; Study design, conceptualization, supervision of lab work, manuscript writing, G K S; Lab work, preparation of manuscript, K V R; Recruitment of subjects, diagnosis and management of cases, M S; Lab work. Conflict of Interest: None. Role of Funding Source: None.
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Indian Journal of Pediatrics, Volume 77—July, 2010