Respiratory Syncitial Virus in Children with Acute Respiratory

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					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,

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:

Key words : Respiratory syncitial virus (RSV); Bronchiolitis; Weight for age; Well nourished children; IL-8

RSV is the most common virus isolated in respiratory                rather than from virus induced cytopathy. 5 Children who
infections1 and is frequently detected in hospitalized              experience RSV infection in early in life, run a high risk of
children.2 It is the main cause of bronchiolitis worldwide          subsequent asthma and recurrent wheezing. Moreover,
and causes up to 70-80% lower respiratory infections                the sera and nasal secretions of the RSV infected infants
during monsoon and winter.3 In India, studies based on              show a marked increase in the levels of Th-2 cytokines
the isolation and sero survey have demonstrated RSV to              and chemokines.6
be the main virus responsible for lower respiratory tract
                                                                       In vitro studies have shown that epithelial cells or
infections in children below 5 yrs of age.4 The severity of
                                                                    macrophages infected with RSV secrete high concentration of
RSV infection in young children varies from a non clinical
                                                                    IL-87. Studies on infants infected with RSV also show elevated
or mild respiratory infection to severe lower respiratory
                                                                    levels of IL-8 in serum and nasopharyngeal aspirate and to the
tract infection that may lead to hospitalization and
                                                                    large number of neutrophils seen in the lavage fluid of
occasionally death. Though, the mechanisms underlying
                                                                    these infants.8 It is now recognized that neutrophils play an
the highly variable disease course in children is still
                                                                    important role in viral respiratory tract infections, but
poorly understood, it is believed that children with severe
                                                                    importance of their role in eliminating viral infection is still
RSV disease suffer from enhanced inflammatory lesions
                                                                    unclear and indeed they may have little or no effect in limiting
                                                                    viral replication. However, there is evidence that they play an
Correspondence and Reprint requests : Dr. R. Hemalatha, MD,         important role in the causation of symptoms during
Deputy Director, Head, Department of Microbiology, National         respiratory viral infections. 8 Several studies have
Institute of Nutrition, Indian Council of Medical Research, Jamai   suggested that IL-2 is an appropriate means of
Osmania (PO), Hyderabad- 50000 7, Andhra Pradesh, India.            determining inflammatory response in patients with
[DOI-10.1007/s12098-010-0108-z]                                     severe acute viral bronchiolitis, in the acute phase of the
[Received July 06, 2009; Accepted April 19, 2010]                   disease.9

Indian Journal of Pediatrics, Volume 77—July, 2010                                                                             755
                                                     R. Hemalatha et al

    The mutually adverse interactions between                    High Performance Liquid chromatography (HPLC).
malnutrition and infection are complex and operate               Anthropometric measurements were taken to assess their
through various mechanisms. The immune response,                 weight for age, using Gomez Classification.
especially cell mediated immunity is known to be poor in         Nasopharyngeal Aspirate (NPA) was collected aseptically
undernourished children. There are, however, very few studies    by passing size 5 feeding tube into the nasopharynx and
to show the relationship between RSV infection, nutritional      applying gentle suction with a syringe. Secretions were
status of the children and local cytokine response which plays   rinsed into collecting vials with 1ml of phosphate buffer.
a key role in the outcome of the disease. The present            After centrifugation of NPA to precipitate cells, the
study was undertaken to investigate the nutritional status       supernatant was frozen at -70 0 until analyzed for
of RSV infected children having respiratory infections,          cytokines and serum samples by ELISA (Diaclone
with special attention to body weight, age, hemoglobin,          Research). Total protein from NPA was determined by
vitamin A and local IL-2 and IL-8 from nasopharyngeal            Modified Lowry’s method. Statistical analysis of the data
aspirate.                                                        was done using SPSS software.

              MATERIALS AND METHODS                                                         RESULTS

The present cohort study was carried out at a tertiary care      Over a period of eight months, a total of 126 children with
centre, Niloufer Hospital, Hospital for Women and Child          acute respiratory infection were recruited. Of the 126
Health, Hyderabad from the months of August through              children recruited, 21 had URTI, 38 had bronchiolitis and
March 2007-2008. Inclusion criteria were: All children           67 had pneumonia. A total of 56 children were positive for
with respiratory infection who had visited the out               RSV while, 70 were negative. Distribution of clinical cases
patient(OP) department during the study period,                  in the age groups <1 yr and ≥1 yr showed a significantly
diagnosis of Pneumonia, bronchiolitis or Upper                   higher proportion of younger infants to be infected with
respiratory infection(URI) with history of illness not more      RSV. The mean age of children with RSV was 8.4 months
than five days. Exclusion criteria were: Children with           and 9.4 months for RSV negative children. Of the 126
congenital heart disease, chronic lung disease, family           cases, 22 children were treated as outpatients, and 104
history of asthma, and other chronic disorders. This study       were hospitalized. Of these 104, 99 children recovered and
was approved by the Institutional Review Board (IRB)             five children (3 RSV + and 2 RSV -) had fatal outcomes
and written informed consent was taken.                          and died in the hospital (Table 1).
   Acute respiratory tract infection was defined as the            Children with bronchiolitis (60.5%) were more commonly
presence of cough with or without fever for less than 2          positive for RSV compared to URTI (19%) and pneumonia
wks. Bronchiolitis was diagnosed in those who presented
with the prodromal symptoms of rhinorrhea, cough, low            TABLE 1. RSV Prevalence and Its Association with Various
grade fever, followed by paroxysmal wheezy cough,                         Parameters
dyspnea, with signs of air hunger, chest retractions,                                       RSV +ve(56)      RSV–ve (70)
wheezing on physical examination and hyperinflation of
lungs with patchy atelectasis on chest radiograph.               Respiratory infection(n)     n (%)           n (%)
Pneumonia was diagnosed in those who presented with                URTI(21)                   4 (19.0)       17 (81.0)
                                                                   Bronchiolitis(38)         23 (60.5)       15 (39.5)
history of poor feeding, respiratory distress with minimal         Pneumonia(67)             29 (43.3)       38 (56.7)
wheeze, cough with chest retractions, shaking chills             Outcome(n)
accompanied with fever, clinical findings of decreased             Recovered(99)             48 (48.5)       51 (51.5)
breath sounds, consolidation or scattered rales heard all          Death(5)                   3 (60)          2 (40)
over the chest over the affected areas, and features               OP(22)                     5 (22)         17 (77.2)
                                                                 Age (n)
suggestive of consolidation or patchy or interstitial
                                                                   <1 yr(89)                 47 (52.8)       42 (47.2)
infiltration on chest radiography.                                 >1 yr(37)                  9 (24.3)       28 (75.7)
   For all the cases, clinical history and physical
                                                                   Males(87)                 37 (42.5)       50 (57.5)
examination was done and recorded. Weight was                      Females(39)               19 (48.7)       20 (51.3)
measured to the nearest 100 g with an electronic scale           Wt/Age (n)
(Secca). Infants were weighed with the help of an                  <75% (71)                 27 (38)         44 (62)
infantometer. Low birth weight was defined as those                >75 %(55)                 29 (52.7)       26 (47.3)
infants who were weighing ≤ 2499 g at birth, irrespective        Birth Weight (n)
                                                                   Low(26)                    9 (34.6)       17 (65.4)
of gestational age. From all cases, 2 ml blood was collected       Normal(94)                47 (50.0)       47 (50.0)
in a plain vial for measuring Hemoglobin (Hb) status and
Vitamin A levels. Vitamin A levels were determined by            n followed by percentage in parenthesis

756                                                                     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                             5000
among boys (42.5%) and girls (48.7%), (P=0.279). A higher                                                            a                                 IL8 1st day
proportion of younger children and well nourished children                                                                           a                 Il8 5th d ay
were positive for RSV (Table 1). The mean WFA and the                                                         a
serum Vitamin A levels were higher in the RSV infected
children, while the hemoglobin status and height for age                                3000
was comparable.
                               a                                          Il2 1st day
                                                                          Il2 5th day
                         250          a                         a

 IL-2 in pg/gm protein


                                                                                                             pneu mo nia            b ro nchio litis

                                                                                                                     clinical diagnosis
                                                                                        Fig. 2. IL-8 levels in children with pneumonia and bronchiolitis
                                                                                                on days 1 and 5.
                          50                                                            Means with different superscript are significantly different with
                               pneumonia              bronchiolitis
                                      clinical diagnosis
Fig. 1. IL-2 levels in children with pneumonia and bronchiolitis
        on days 1 and 5.                                                                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
    In a sub sample of 18 RSV positive and 7 RSV negative
                                                                                        children with bronchiolitis or pneumonia, RSV has been
cases, nasopharyngeal IL-2 and IL-8 was done. The total
                                                                                        found to be associated with a higher proportion of
concentration of IL-2 and IL-8 ranged from 45.4 to 722 pg/g
                                                                                        bronchiolitis compared to pneumonia.10 Similarly in the
total protein and 597 to 8127 pg/g total protein, respectively,
                                                                                        present study, RSV was more commonly associated with
with IL-2 being detectable in less than 50% of the cases( 12 out
                                                                                        bronchiolitis than pneumonia or upper respiratory
of 25). However, after 5 days, IL-2 was detectable in nearly all
                                                                                        infections (URTI). Earlier studies showed that RSV
cases (22 out of 25 cases), but the total concentration decreased
                                                                                        prevalence was found to be more in males than females11
from the initial level. The mean concentration of IL-2 and IL-8
                                                                                        and male gender was considered an independent risk
were higher in the RSV positive cases though not statistically
                                                                                        factor for the development of severe RSV infection. In
significant (Table 2). Categorizing the cases based on their
                                                                                        contrast, the authors observed an equal distribution of
clinical outcome, it was found that on day 1, bronchiolitis
                                                                                        RSV in boys and girls though association with younger
and pneumonia cases had high IL-2 and IL-8 levels,
                                                                                        age was similar to that reported elsewhere. 12 Though
however, children with bronchiolitis had relatively lower
                                                                                        malnutrition has been found to be a risk factor for acute
concentrations of IL-2 compared to children with pneumonia
                                                                                        LRI, 13 animal studies on RSV infection and nutrition
(Fig. 1). On day 5, children with pneumonia maintained same
                                                                                        showed conflicting results.14 In the present study, good
concentrations of IL-2 and IL-8 while children with bronchiolitis
                                                                                        nutritional status (WFA) and higher Vitamin A levels
secreted lower IL-8 levels (Fig 2). Children with pneumonia,
                                                                                        were associated with RSV infection. A similar observation
positive for RSV had significantly higher IL-8 concentrations
                                                                                        was made by Clarios et al who reported good nutrition,
compared to those without RSV infection.
                                                                                        obesity and over nutrition as risk factors for RSV
TABLE 2. RSV Infection and IL2, IL8 Response in Children with
         ARI                                                                               The role of inflammatory mediators in the
                                                                                        pathogenesis of RSV disease though not well-understood,
                                          RSV +ve                       RSV -ve         has been studied by various people. In vitro studies have
Vit A(µg/dl)                        19.95   ± 1.513                    16.42 ± 1.769    shown that epithelial cells or macrophages infected with
Hb (g/dl)                           11.01   ± 0.298                    10.78 ± 0.237    RSV secrete high concentration of IL-8.7,15,16 Similar to the
IL-2   day 1                          285   ± 64.76                    235.3 ± 29.18    present study, other studies on infants infected with RSV
       day 5                          213   ± 44.4                     157.7 ± 33.21    also showed elevated levels of IL-8 in serum and
IL-8   day 1                       3425.2   ± 436.5                   2176.7 ± 666.84
                                                                                        nasopharyngeal aspirate that has been suggested in
       day 5                       2670.9   ± 384.8                   1092.6 ± 180.5
                                                                                        contributing to large number of neutrophils recruited in
IL-2 and IL-8: pg/gm of total protein. The values are Mean ± SE                         the lavage fluid of these infants. 8 That neutrophils

Indian Journal of Pediatrics, Volume 77—July, 2010                                                                                                                    757
                                                          R. Hemalatha et al

recruited by local IL-8 secretion might contribute to the              5. McNamara PS, Smyth RL. The pathogenesis of
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while some investigators suggested IL-8 polymorphism in                   childhood. Pediatr Allergy Immunol 2002; 13: 47-50.
the pathogenesis of hypersensivity reaction, others                    7. Becker S, Quay J, Soukup J. Cytokine (TNF, IL-6, IL-8)
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                                                                       9. Katia M, Giugno, Denise C et al. Concentrations of IL-2 in the
the airway inflammation that results from the viral                       nasopharyngeal secretions of children with acute respiratory
infection is associated with a marked increase in IL-8 in                 syncitial virus bronchiolitis. Journal de Pediatr 2004; 80(4)
the infected sites of the lung, which might contribute to             10. Parrot RH et al. Epidemiology of RSV infection in Washington
increased local leukocyte response and hypersensitivity                   DC-Infection and disease with respect to age, immunological
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                                                                      11. Rivera Claros R, Marin V, Castillo-Duran C, Jara L, Guardio S.
factor for RSV infection, however, it does not seem to                    Nutritional status and clinical evaluation of hospitalized
decrease the chances of these children developing                         Chilean infants with infection by RSV. Arch Latinoam Nutr
sequelae at a later date.                                                 1999; 49: 326-332.
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Acknowledgements                                                          RSV lower respiratory tract disease. J Pediatr 2003; 143: S118-
We are grateful for the skillful assistance rendered by the nursing       S126.
staff Santoshamma and Rajakumari in sample collection at Niloufer     13. Savitha MR, Nandeeshwara SB, Pradeep Kumar MJ, Ul-
hospital.                                                                 Haque F, Raju CK. Modifiable risk factors fro acute lower RTI.
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Contributions: R H; Study design, conceptualization, supervision of   14. Victor Pena Cruz, Carol R, McIntosh Kenneth. Effect of RSV
lab work, manuscript writing, G K S; Lab work, preparation of             infection on mice with protein malnutrition. J Med Virol 1991;
manuscript, K V R; Recruitment of subjects, diagnosis and                 33: 219-223.
management of cases, M S; Lab work.                                   15. Becker S, Koren HS, Henke DC. Interleukin-8 expression in
                                                                          normal basal epithelium and its modulation by infection with
Conflict of Interest: None.
                                                                          RSV and cytokines TNF, IL-1, IL-6. Am J Respir Cell Mol Biol
Role of Funding Source: None.                                             1993; 8: 20-27.
                                                                      16. Arnold R, Humbert B, Werchau H, Gallati H, Konig W. IL-8,
                                                                          Il-6 and soluble TNF receptor type 1 release from a human
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