Acute respiratory tract infections play a major role in hospitalizations of children, and respiratory syncytial virus is well recognized as the most important pathogen causing ARTI.Objectives: This study aimed to evaluate epidemiological and clinical patterns of RSV infection in children hospitalized for lower ARTI in Ahvaz, Iran.
Patient and Methods: Respiratory specimens collected from 100 children with lower ARTI from October 2008 until the end of April 2009, were screened for RSV using real-time reverse transcription-polymerase chain reaction (RT-PCR).
Results: During the study period, 9 children had a positive result for RSV infection. The median age of these patients was 10 months. Bronchiolitis was the clinical diagnosis of patients with RSV infections. All of these patients were less than 24 months. Cough (77.7%) and chest wall retraction (100%) were the leading symptoms and signs respectively.Conclusions: This study indicates that RSV is an important cause of respiratory tract infection in infants less than 2 years old. RT-PCR offers a rapid method for common respiratory viruses.
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Key words: Children, lower respiratory tract infection, Respiratory syncytial virus, Rt-PCR.
Acute respiratory tract infections (ARTIs)are a leading cause of hospitalization and
mortality in children less than 5 years of age and represent a considerable health problem in the world.(1) Viruses, including respiratory syncytial virus (RSV), metapneumovirus, influenza A and B, parainfluenza and adenovirus are the most common etiological agents for childhood acute respiratory tract illness. (2) Among viral causes of acute respiratory tract infections, RSV has a significant role. Human respiratory syncytial virus is an enveloped, single-stranded; negative-
sense RNA virus of the genus Pneumovirus.The peak incidence of the RSV infections is between the second and six month of age. It can cause significant morbidity from upper respiratory infections, acute bronchiolitis, and bronchopneumonia to apnea in children. (3, 4)In hospitalized children, RSV infections occur at greater frequency than other viral
infections of the lower respiratory tract. It is identified as the etiologic agent in 60-90% of the patients with bronchiolitis and in 25-50% of the pneumonia. (5, 6)
In temperate countries, RSV outbreaks have a defined seasonality, occurring mainly fall
and winter while in tropical and semi-tropical countries, it mainly peaks during the rainy season.(7, 8) In addition to conventional viral culture techniques and serology, a recently described innovative, polymerase chain reaction (PCR) for the diagnosis of respiratory viral infections has also been shown to be useful because it offers an enhanced sensitivity combined with rapid detection. Even if certain viruses such as RSV can be grown in cell cultures, this method is not 4 completely reliable and many scientists have begun to use real-time polymerase chain reaction
RT-PCR to identify infections.(9,10)Information on the epidemiology of RSV infections in developing countries are still limited. The most of these studies have not used highly sensitive molecular techniques such as(RT-PCR).(11-13) 2.Objectives
The purpose of this study was to provide data about the detection of RSV by RT-PCR in
hospitalized children up to age of five years in Ahvaz, Iran.
3.Patients and Methods
The study population consisted of children less than 5 year of age, admitted in the
Aboozar children's hospital with lower respiratory tract infections (LRTI). It was designed to enrolled subjects from the beginning of October 2008 until the end of April 2009.Newborn infants less than 28 days were excluded.LRTI were categorized on the basis of clinical and roentgenographic findings as pneumonia and bronchiolitis. Bronchiolitis was defined as an acute respiratory illness characterized by
rhinorrhea, cough, and dyspnea and diffuses wheezing, with peribronchial thickening and hyperexpansion on chest radiograph if available. Pneumonia was defined as dyspnea in a patient with focal rales or decreased breathing sounds and the presence of a focal infiltrate and/ or consolidation on chest radiograph.5 During hospitalization, the children's signs and symptoms were obtained by a review of medical charts. This information was reviewed and entered into a database. Informed consent was
obtained from the parents.Following the clinical assessment, nasopharyngeal specimens were collected by gently rubbing the deep nasal turbinate with Dacron swabs. They were kept in viral transport medium and were stored at -70° until further testing in the virology laboratory of infectious diseases research center. Samples were obtained within 24 hours after admission.RT-PCR Initially RSV RNA was extracted from nasal sample by using high pure RNA nucleicacid kit (Roche company), followed by cDNA preparation for the each sample. The Nested PCR was carried out and following primers were used. (Johnson)
Always on Time
Marked to Standard
G1- CCA TTC TGG CAA TGA TAA TCT C
G2- GTT TTT TGT TTG GTA TTC TTT TGC GA
G3- CGG CAA ACC ACA AAG TCA CAC
G4- GGG TAC AAA GTT AAA CAC TTC
The primers G1 and G2 were used for the first round. The 25 µl of PCR master mix
containing 5 µl of the cDNA of the each sample, 12.5 of the 1- PCR master mix, 50 pmol of the each G1 and G2 primer was added to master mix. The PCR was performed for 40 cycles in Techne Thermocycler UK, consisting initially 5 min for 95°C and finally 5 min at 72°C for one cycle. The primers G3 and G4 were used for the second round. The 25 µl of PCR master mix containing 5 µl of the cDNA of the each sample, 12.5 of the 1-PCR master mix, 50 pmol of the 6 each G3 and G4 primer was added to master mix. The PCR was performed for 35 cycles consisting initially 5 min for 95°C followed by, 1 min at 72°C, 1 min at 95°C and finally 5 min at 72°C for one cycle. The expected final PCR product was 326 bp.This study was approved by the Ethics Committee of Ahvaz Jundishapur University ofMedical Sciences.
One hundred specimens from hospitalized children with LRTI were tested for a possible
RSV infection .There were 57 males (57%) and 43 (43%) females. The median age of patients at the time of study was 21months and 63 patients (63%) were younger than 2 years. (Table 1)The clinical diagnoses at the time of admission and discharge were as follows:pneumonia 64 (64%) and bronchiolitis 36 (36%).
The overall frequency for RSV infection among the 100 children younger than 5years of
ages admitted to Aboozar children hospital was 9%. The median age of RSV-infected children
was 8.5 months. Bronchiolitis was the clinical diagnosis of patients with RSV infections.All of the patients with RSV infections was less than 2 years old.
Cough was the leading symptoms (77.7%) followed by coryza (66.6 %) and fever (55.5%). Evidence of lower respiratory tract infection were chest wall retraction (100%), wheezing(88.8%) and cyanosis (22.2%).(Table 2)The clinical outcomes for children infected with RSV were good in general.
Viral infections are considered the most important cause of lower respiratory tract
infections (LRTI). It is responsible for a significant mortality and morbidity in children. RSV is one of the most frequent etiological agents causing LRTI, especially among young infants. (14,15)Lower respiratory tract infection (LRTI) is responsible for a significant mortality and morbidity in children. RSV is recognized to be a major viral cause of LRTI, especially among young infants worldwide.RSV epidemics occur yearly but may alternate in occurrence between midwinter and early spring. Its distribution varies in different countries and seasons. (7) During this study RSV was detected in 9% of all subjects included .The percentage of laboratory-confirmed RSV cases in Greece, Jordan and Bulgaria were 5.4%, 12.5% and 8.46% respectively, that are comparable with our study. (1, 16, 18)The prevalence of RSV in this study is relatively low compared with data reported in northern European and American countries. (10, 14, 15, 19)
Other studies from Iran have showed incidences of RSV infection 12.9%, 15%, 16.8 % in cases of LRTI and all of these reports are from temperate regions. (20-22)
The difference of RSV epidemiology in the world may be related to differences in
climate conditions, in environmental factors and in severity of its epidemics from one year to another.(10)It has reported negative correlations between higher monthly mean temperature and RSV infection in tropical regions. 21 Ahvaz is in the southwest of Iran and has fall-winter seasons characterized by relatively warm not favoring the survival and the spreading of RSV.8 Studies have shown that some pediatric patients with acute lower respiratory tract infection become infected simultaneously with multiple respiratory viruses. Dan peng et al suggests that there is a high occurrence of multipathogen infections in children admitted withacute respiratory tract infections and that coinfection is associated with certain pathogens. In this study RSV was detected in only 5.7% of cases.(23)In addition, Arabpour et al reported high prevalence (54.4%) of Human
metapneumovirus (hMPV), among Ahvazian children with respiratory tract infections. This study showed the probable role of hMPV as an important causative agent of acute respiratory tract infections in this area. (24) In our survey, there was a slightly higher incidence of RSV infection in males (1.3:1).This result was consistent with other studies. Sangar et al suggested that being female was protective against RSV hospitalization.(25,26)In this study all of the children that tested positive for RSV had bronchiolitis and the highest number of positive samples for RSV is in the patients less than 1 year.This tendency toward younger age in RSV infections has been reported in other reported data.(27,28)The clinical features of children with RSV positive samples observed in our study were similar to those of previous reports. (14, 15)
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There were two major limitations in our study. One of our limitation is though some
children presented the criteria for inclusion in this study, samples from 100 patients were
collected, due to the limit established for processing and storage of samples. The second limitation is we did not check out all of the respiratory viral agents in our patients. It seems in 9 our geographic position and climate situation the other viruses like hMPV have an important role in LRTI in children.Infections with respiratory viruses are a common cause of morbidity and mortality around the world. A better understanding of the epidemiology of respiratory viral infections may be used for timely, specific antiviral therapy, prophylaxis, and vaccination. Future prospective surveillance over an extended period in this region is needed to accurately identify the epidemiology of viruses.