A Prospective Three-Year Cohort Study of the Epidemiology and Virology of Acute Respiratory Infections of Children in Rural India

Background Acute respiratory infection (ARI) is a major killer of children in developing countries. Although the frequency of ARI is similar in both developed and developing countries, mortality due to ARI is 10–50 times higher in developing countries. Viruses are common causes of ARI among such children, yet the disease burden of these infections in rural communities is unknown. Methodology/Principal Findings A prospective longitudinal study was carried out in children enrolled from two rural Indian villages at birth and followed weekly for the development of ARI, classified as upper respiratory infection, acute lower respiratory infection (ALRI), or severe ALRI. Respiratory syncytial virus (RSV), influenza, parainfluenza viruses and adenoviruses in nasopharyngeal aspirates were detected by direct fluorescent antibody testing (DFA) and, in addition, centrifugation enhanced culture for RSV was done. 281 infants enrolled in 39 months and followed until 42 months. During 440 child years of follow-up there were 1307 ARIs, including 236 ALRIs and 19 severe ALRIs. Virus specific incidence rates per 1000 child years for RSV were total ARI 234, ALRI 39, and severe ALRI 9; for influenza A total ARI 141, ALRI 39; for INF B total ARI 37; for PIV1 total ARI 23, for PIV2 total ARI 28, ALRI 5; for parainfluenza virus 3 total ARI 229, ALRI 48, and severe ALRI 5 and for adenovirus total ARI 18, ALRI 5. Repeat infections with RSV were seen in 18 children. Conclusions/Significance RSV, influenza A and parainfluenza virus 3 were important causes of ARI among children in rural communities in India. These data will be useful for vaccine design, development and implementation purposes.


INTRODUCTION
Acute respiratory infections (ARI) result in ,1.9 million childhood deaths per year in developing countries, 20% of these deaths are in India [1,2]. The role of viruses in the etiology of ARI in developing countries like India is not well studied. Among children hospitalized with acute lower respiratory infections (ALRI) in India, one half the infections are of viral etiology [3][4][5].
There have been no reports of longitudinal community based studies of viral ARI in India in the past 30 years [6]. The present study was undertaken to describe the epidemiology of ARI due to viruses among young children in two rural villages in India. The development of a capacity for prospective longitudinal studies of ARI in rural India should serve as a valuable resource of investigations of ARI, risk factors for ARI, and evaluation of interventions to reduce ALRI disease burden.

Study Population
Ballabgarh is a rural region in Haryana state in north India near Delhi. The population of over 70,000 is served by the Ballabgarh Primary Health Centre (PHC) under the Comprehensive Rural Health Services Project of All India Institute of Medical Sciences (AIIMS). The computerized records of all families in the communities are available at the PHC. For this study newborns were enrolled from two villages in Ballabgarh, Nwadha and Mujeri, with approximate populations of 2,300 and 1,700 respectively and birth rates of approximately 25 and 26/1000 respectively. The infant mortality rate for Ballabgarh was about 37 per thousand live births in 1997 [7]. The population of the villages is generally stable with very little migration. Newborns were identified from records available at the PHC and parents were asked to enroll their infants in the study. Informed consent was obtained from the parents in Hindi. Institutional review boards in India (Ethics Committee at All India Institute of Medical Sciences) and the USA (Institutional Review Board for human use [IRB] at University of Alabama at Birmingham) approved the study.
Study follow-up and data collection All information obtained was entered onto standardized proformas. Demographic data was collected by an interview with the parents and included child and birth characteristics; education and occupations of parents; and income and material possessions. Prematurity was defined as birth at less than 37 completed weeks of gestation [8].

Monitoring for ARI
The fieldworkers visited the households weekly to identify ARIs in enrollees until the children were 3 years of age or the study ended. The field workers were trained to use WHO definitions for the classification of ARI [9]. Medical officers (physicians) supervised and provided ongoing training of the field workers.

Definitions and Management of ARI
ARI was defined as cough or difficult breathing within the previous 7 days or observed during the visit. Each episode of ARI was considered to last two weeks, a new episode was counted if new signs and symptoms develop after the child has been symptom free for at least one week. The medical officers examined children with ARI/ ALRI. Age adjusted definitions of fast breathing were as follows: age less than 2 months, 60 breaths per minute or more; age 2 months up to 12 months, 50 breaths per minute or more; and age 12 months up to 3 years, 40 breaths per minute or more. Danger signs (the presence of any of these signs required urgent referral to the hospital) were: inability to drink or breastfeed, child vomits everything, child has had convulsions, or child is lethargic or unconscious [9].
Children 2 months to 3 years of age Chest indrawing or stridor in calm child or a child exhibiting any general danger sign was classified as severe ALRI or very severe disease. Treatment included giving the first dose of an appropriate antibiotic if available and referring the child urgently to the hospital in Ballabgarh. Fast breathing was classified as ALRI. Treatment included giving the appropriate oral antibiotic for 5 days and advising the mother as to when to go to clinic immediately. If there were no signs of ALRI or severe ALRI, classification was URI.
Children less than 2 months of age Any general danger sign or fast breathing, chest indrawing, nasal flaring or grunting; classification severe ALRI or very severe disease. These children were referred urgently to the hospital. If these signs were not present classified as URI.

Specimen Collection
At each ARI, a nasopharyngeal aspirate (NPA) was obtained by the medical officer. Specimens were collected through an infant feeding tube placed in the posterior nares and into a mucous trap using suction from a hand vacuum pump (Nalge Nunc Inter., Rochester, NY, USA). Cold viral transport media was aspirated into the mucous trap containing the NPA, the sample was placed on ice and transported to the Virology Laboratory at AIIMS within 6 hours [5].

Virus Identification
Samples were processed and viruses were identified using antigen detection [10]. b-mercaptoethanol (100 ml of 2%) was added and the sample was mixed. Centrifugation was performed and the pellet was washed and resuspended in phosphate buffered saline. Smears from the cell suspension were placed on Teflon coated slides for DFA. Initial screening for presence of viral antigens used the SimulFluorH Respiratory Screen (Chemicon International, Inc.; Temecula, CA, USA). If RSV antigens were present golden fluorescence was observed, with other respiratory viruses (influenza A or B, parainfluenza 1, 2, 3 or adenovirus) green fluorescence resulted [11]. Samples positive by SimulFluor were tested by DFA Kit (Chemicon International) using individual monoclonal antibodies against RSV, influenza A (INF A) and B (INF B), parainfluenza 1 (PIV 1), 2 (PIV 2), and 3 (PIV 3), and adenovirus. Positive and negative controls were included. Positive identification by DFA required positive and congruent results by both SimulFluor and individual monoclonal testing by DFA. All slides with positive immunofluorescence results were reviewed by the senior investigator in charge of the Diagnostic Virology Laboratory at AIIMS (SB). Supernatants from the clarified samples were used for isolation of RSV by CEC [5] and presence of RSV was detected by indirect immunofluorescence staining with a blend of monoclonal antibodies that detects both group A and group B RSV (Chemicon International). Cultures were not performed for other viruses.

Data entry and Analysis
The data for the study were entered into an Epi Info 2000 software database with customized views containing suitable range and consistency checks to prevent invalid data from being entered. All data were double keyed into the database. The overall incidence rates (IR) of ARI, ALRI and severe ALRI were determined by dividing the sum of new episodes by the total number of child years at risk and presented as cases per 1000 child-years. Child years were calculated from the date of enrollment until either the child died, refused further follow up, reached 3 years of age, or through March 30, 2005. Confidence intervals (95%) for IRs were calculated by exact binomial methods.

Characteristics of study population
Population characteristics were similar between the villages Nwadha and Mujeri and the composite data are presented. All of the mothers were married and indicated their occupation as housewife. Many of the mothers were illiterate (46%), their age was 2363.8 years (mean6SD). Fathers were employed as day laborers (21%), farmers (26%) small business owners (25%), or were employees of business or government (28%). Reported median incomes were 36,000 (range 5,000-144,000) rupees (,USD $777, range $108-$3,107) per year. Births were almost equally divided between home (47%) and in a hospital or clinic (53%) and 11% were preterm.

Enrollments in the study
The enrollment began October 1, 2001 and ended after 39 months (December 31, 2004), follow-up concluded after a total study period of 42 months (March 31, 2005). Enrollment was almost equally divided between the two villages (Nwadha 145, Mujheri 136). A total of 281 infants were enrolled, including 148 males and 133 females accounting for 86% of the 325 births in this period. Twenty-two children enrolled and then opted out of the study and six deaths occurred, none were due to ARI. The age at enrollment ranged from 1 day to 10.7 months (mean6SD, 1.461.7) with a median of 0.8 months. The period of follow up ranged from 1 month to 36 months (mean6SD, 20.4610.4) and a median of 20 months. Accounting for deaths and drop-outs there were 440 child-years of follow-up.
Incidence rates by disease category and age are given (Table 1), there were 2966 ARIs, including 536 ALRI and 43 severe ALRI per 1000 child years (cy). By age group severe ALRI was most common in infants (62 per 1000 cy) and 85% of these occurred in infants less than 6 months of age. The rates for ALRI were highest in the 12-23 month age group at 831 per 1000 cy, as compared to 389 for 24-35 months and 370 for 0-11 months. Of the 281 children enrolled 268 (95%) had between one and 15 episodes of ARI.

Virus identification by direct fluorescence assay
Nine hundred forty four samples were sufficient for analysis (adequate cell numbers and not obscured by mucus) by DFA of 1279 NPAs collected. A total of 233 respiratory viruses were identified by DFA ( Centrifugation enhanced culture identification of RSV RSV was detected in 95 of 1279 samples tested by CEC (7.4%). One hundred and three (8%) samples were positive for RSV by one or both assays (DFA and CEC). In 944 samples tested by both DFA and CEC, RSV was detected in 25 (2.5%) by DFA and 75 (7.9%) by CEC. Of these, 13 were positive by both assays, 11 by DFA alone and 62 by CEC alone. Of the 86 samples positive for RSV by one or both assays 87% (75/86) were detected by CEC and 28% (24/86) by DFA. Thus, sensitivity of RSV detection was increased by use of both the techniques, DFA and CEC [5]. Some of the RSV strains underwent molecular characterization [12,13].

Distribution of viruses by age and severity of illness
RSV, INF A, and PIV 3 infection incidence rates (IR) are shown by disease category and age ( Table 3). The RSV total ARI IR was highest in the first year of life (322 per 1000 cy). The RSV associated ALRI IR was actually higher in the 12-23 months group as compared to other ages, whereas for RSV severe ALRI the IR was slightly higher in the 0-11 month as compared to the 12-23 month group. RSV was found in 8% of URIs, 7% of ALRIs, and 21% of severe ALRIs. RSV was identified by DFA and CEC, for the other viruses only DFA was performed (944). Thus, IRs cannot be directly compared between RSV and the other virus types. INF A total ARI IR was 141 per 1000 cy, the IR for ALRI was 39, and no severe ALRIs were identified. INF A IR were similar for 0-11 and 12-23 months and slightly lower in the 24-35 month group. INF A was found in 6% of URIs and 11% of ALRIs. The PIV3 total ARI IR was 229 per 1000 cy, with ALRI 48 and severe ALRI 5 per 1000 cy. In the first 2 years of life the IR for PIV3 was very similar, but fell in the third year. PIV 3 was found in 10% of URIs, 11% of ALRIs, and 15% of severe ALRIs.
INF B, PIV1 and 2, and adenovirus are not shown because they were identified rarely in children with ALRI and never from children with severe ALRI. The total IRs were as follows: INF B 37, PIV1 23, PIV2 28, and adenovirus 18 per 1000 cy. INF B and PIV1 were not identified in ALRIs. The IR for ALRI for PIV 2 was 5 and for adenovirus was 5 per 1000 cy.

Monthly distribution of viruses
The monthly distribution of viruses is shown (Figure 1). RSV identifications were most frequent in the fall and winter but continued at a lower level throughout the year with the exception of the rainy season of July and August. During the fall and winter 2004-2005 much more PIV 3 and INF A activity was noted as compared to the previous years and these viruses were found more often than RSV in this epidemic period.

Repeat Infections with RSV
RSV primary infections were found in 30% (83/281) and 18 children (6.4%) had one or more reinfections, 14 children with one and 4 with two documented reinfections (  Most virus identifications occurred in the fall and winter as expected from a temperate climate [5]. A 1964-1969 study in rural villages outside tropical Calcutta found RSV primarily during September and October, following the monsoon season [23]. The same group found PIV identifications exceeded that of RSV, with PIV in 11% and RSV in 2% of children in the years 1964-1966 [6]. In the current study the 2004-2005 epidemic period was notable for the contribution of both INF A and PIV 3 to the viral ARI disease burden. Reinfections with RSV are the norm and occur throughout life. In this study the IR for reinfections was lower (50 per 1000 cy) than that reported from Kenya (192 per 1000 cy) [20]. In 3 children repeat infections were ALRI, thus primary infection did not protect against later lower respiratory disease. This is in agreement with earlier work that found a single infection may not be sufficient to reduce illness upon reinfections [24]. A subset of the viruses presented here underwent detailed molecular characterization [13]. In this study and in work done in Kenya viruses belonging to the same genetic groups or lineage or even apparently identical viruses have been detected in reinfections [25]. The role of antigenic diversity in the establishment of reinfections requires further investigation. This is the first community-based study of viral ALRI in India in more than three decades [6]. The results show that RSV, INF A, and PIV3 are important causes of ARI, including ALRI, among young children. Severe ALRI was more commonly caused by RSV than by INF A or PIV 3. Because standard WHO definitions were used and a stable cohort was followed prospectively these data include denominator information typically unavailable from hospital-based studies. These data will be useful for planning the study of future respiratory virus vaccines or other interventions to reduce the disease due to viral ALRIs. However, additional studies with a larger population will be required to more precisely define viral ARI disease burden.