|Year : 2012 | Volume
| Issue : 2 | Page : 155-158
Prevalence of influenza virus among the paediatric population in Mumbai during 2007-2009
S Roy1, D Patil1, R Dahake1, S Mukherjee1, SV Athlekar2, RA Deshmukh1, A Chowdhary1
1 Department of Virology, Haffkine Institute, Mumbai 400 012, India
2 Department of Clinical Pathology, Haffkine Institute, Mumbai 400 012, India
|Date of Submission||11-Jan-2012|
|Date of Acceptance||17-Feb-2012|
|Date of Web Publication||28-May-2012|
Department of Virology, Haffkine Institute, Mumbai 400 012
Purpose: Influenza has a major impact on public heath, annually affecting 15-20% of the global population. Information on the activity of influenza virus in Mumbai is limited. The present study was carried out to determine the prevalence of influenza viruses causing acute respiratory infections in children by molecular methods. Objective: To study the prevalence of influenza viruses among the paediatric population in Mumbai by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). Materials and Methods: From July 2007 to July 2009, 100 respiratory samples (nasal and throat swabs) were collected from paediatric patients with acute respiratory symptoms. attending out patients department, and admitted to the paediatric wards of B. J. Wadia Hospital for Children, Mumbai. The samples were collected and processed as per World Health Organization (WHO) guidelines. Viral RNA was extracted and one-step rRT-PCR was performed to detect influenza type A (H1 and H3) and influenza type B virus. Results: Out of 100 samples processed by rRT-PCR, a total of 11 samples (11%) were positive for influenza virus. The typing for influenza A subtypes showed 1% (1) positivity for H1 and 5% (5) positivity for H3 subtypes and 5% (5) samples tested positive for influenza type B virus. Conclusion: It was observed that both influenza type A and B viruses were prevalent in Mumbai during the study period. Such surveillance data are important in the early detection of any antigenic variants that may be helpful in global influenza vaccine preparation and for any pandemic preparedness activity.
Keywords: Influenza virus, paediatrics, prevalence, rRT-PCR
|How to cite this article:|
Roy S, Patil D, Dahake R, Mukherjee S, Athlekar S V, Deshmukh R A, Chowdhary A. Prevalence of influenza virus among the paediatric population in Mumbai during 2007-2009. Indian J Med Microbiol 2012;30:155-8
|How to cite this URL:|
Roy S, Patil D, Dahake R, Mukherjee S, Athlekar S V, Deshmukh R A, Chowdhary A. Prevalence of influenza virus among the paediatric population in Mumbai during 2007-2009. Indian J Med Microbiol [serial online] 2012 [cited 2013 May 18];30:155-8. Available from: http://www.ijmm.org/text.asp?2012/30/2/155/96670
| ~ Introduction|| |
Acute respiratory tract infections (ARIs) are a leading cause of morbidity and mortality in children worldwide,  accounting for about 30% of all childhood deaths in the developing world.  One-quarter (2.5 million) of the total deaths among children less than 5 years of age occurs in India and approximately 20% of these are due to ARIs. Respiratory viral infections have a major impact on public health. Of all the viruses that cause respiratory illness, influenza virus plays a dominant role. Influenza viruses are known to cause frequent epidemics and periodic pandemics, and are unique with regard to their antigenic variability, seasonality and impact on general population. Though children are mainly affected during epidemics,  the viruses are also responsible for substantial mortality in the aged and chronically ill persons.  Studies on the aetiology of ARIs have been carried out in many parts of India. , Influenza like illness (ILI) screening and surveillance programme are critical in tracking the activity, especially of influenza viruses, across seasons. Since the critical differential diagnosis is difficult, such programmes become imperative in the control and management of respiratory viral disease outbreaks. Haffkine Institute is a World Health Organization (WHO) Influenza Surveillance centre and has been engaged in surveillance of influenza virus in Mumbai region. Since very little information on influenza virus activity and prevalence in Mumbai is available, the present study was carried out to determine the prevalence of influenza viruses in paediatric population, both in hospitalised patients and from the outpatient department of B. J. Wadia Children Hospital, Mumbai.
| ~ Materials and Methods|| |
A total of 100 clinical samples were collected from children in the age group of 5 months to 12 years, from the outdoor patient department (OPD) of B. J. Wadia Hospital for Children, Mumbai, showing characteristic ILI, during July 2007 to July 2009. Due permission from the Institutional Ethics Committee of Haffkine Institute was obtained vide letter No. HITRT/MO/IEC 4368 dated 5 June 2007. Clinical samples collected in a viral transport medium (VTM), in a triple layer packing, were transported in a Styrofoam box containing ice packs or in a vaccine carrier box to the laboratory. The samples were processed in a Biosafety level II Cabinet and divided into three aliquots and stored at -80°C in deep freezer. , The viral RNA was extracted from clinical samples using the spin column based QAIamp® Viral RNA mini kit (Qiagen GmbH, Hilden, Germany) as per the manufacturer's instructions. Primers and probes were custom synthesised from Applied Biosystems (ABI), Ambion, USA, for influenza type A, M (Matrix) gene, H1 and H3 gene, and influenza type B, NS1 gene sequences, as shown in [Table 1].  Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) was performed by using a StepOne Real Time PCR instrument (ABI) with an rRT-PCR kit containing 2 × PCR mix and SuperScript™ III RT/Platinum Taq Mix (Enzyme mix) from Invitrogen (CA, USA), and influenza A (H1), (H3) and influenza B primers and probes. A master mix of 20.0 μL was prepared in a PCR plate and 5 μL of RNA template was added and the plates were placed in the ABI StepOne Real-time PCR instrument using cycling conditions of 50°C for 30 min of reverse transcription followed by Taq inhibitor inactivation at 95°C for 10 min and PCR amplification (45 cycles) at 95°C for 15 sec and at 55°C for 30 sec.  Chi-square test and two-tailed Z-test were applied to determine the associations between influenza positivity and demographic/epidemiological data.
|Table 1: Infl uenza type A (H1) and (H3) and infl uenza type B RNaseP primer and probe sequence|
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| ~ Results|| |
From the case history and demographic data, it was found that out of 100 clinical samples collected, 57 (57%) were male patients and 43 (43%) were female patients showing ILI symptoms. Population between 1 and 5 years was found to be majorly affected, which formed 73% [Figure 1].
|Figure 1: Percentage of age distribution in paediatric population diagnosed as infl uenza|
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It was found that 12.28% (7/57) of influenza-positive samples were males and 9.30% (4/43) were females; however, statistically there was no significant difference observed with respect to influenza positivity in gender-wise distribution. Symptomatically, fever (83%), chills and rigor (14%), nasal discharge (83%), ear discharge (8%), cough (86%), sore throat (35%), breathlessness (35%), headache (17%), body ache (17%), fatigue (11%), ARI in family (6%), vomiting (13%) and diarrhoea (3%) were the symptoms observed in ILI patients. Of these, 2% of patients had visited outside Mumbai and none were immunised and taken antibiotic treatment.
In clinical presentation of positive influenza cases, nasal discharge was prominent in every patient. Symptomatically, nasal discharge (100%), cough (91%), fever (73%), breathlessness (36%), sore throat (27%), headache (27%), body ache (27%), chills and rigor (9%), ear discharge (9%), fatigue (9%), vomiting (9%), diarrhoea (9%) and ARI in family (9%) were the clinical presentations observed in influenza-positive population [Figure 2].
|Figure 2: Clinical presentation of laboratory confi rmedinfl uenza- cases|
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Out of 100 samples collected from ILI patients from B. J. Wadia Hospital for Children, 11 (11%) samples were positive for influenza virus. Of these positive samples, 1 (1%) and 5 (5%) were positive for influenza type A H1 and H3, respectively, and 5 (5%) samples were positive for influenza type B virus by rRT-PCR method [Figure 3]. Statistical tests applied showed no significant association between influenza positivity and demographic/epidemiological data.
| ~ Discussion|| |
The incidence of influenza in children is difficult to estimate because most children with influenza have few or mild symptoms or none at all. However, children at school are central to the spread of influenza in the community. Trivalent inactivated vaccines are currently used as preventive measures, but a trivalent attenuated cold-adapted vaccine might be more efficient and acceptable for children. Because clinical diagnosis alone is unreliable, especially in young children, laboratory confirmation is needed. To obtain more detailed information about the prevalence and impact of influenza, the prevalence of influenza in Mumbai was studied. Children aged <15 years with an ILI enrolled in the influenza surveillance study had high rates of hospitalisation and outpatient morbidity when influenza virus was circulating in the community.
Of the 100 clinical samples collected from patients with ILI symptoms, the patient distribution was observed to be 57% males and 43% females, giving a male: female sex ratio of 1:0.754. It was observed that gender-wise positivity for influenza virus in male and female was 12.28% (7/57) and 9.30% (4/43), respectively; however, statistically there was no significant difference with respect to influenza positivity in gender-wise distribution on using two-tailed test for proportion. In clinical presentation of positive influenza cases, nasal discharge was prominent in every patient. Cough was found in 10 cases (91%), fever in 8 cases (73%), breathlessness in 4 cases (36%), and sore throat, headache and body ache were found in 3 cases (27%). In subtype-specific positive influenza cases, nasal discharge (100%) was the most common finding in all subtypes of influenza. Although influenza is principally a respiratory infection, it was surprising that only 36% of the children were admitted mainly for breathlessness. This finding is important, as it could be assumed that children with primarily non-respiratory clinical manifestations may easily remain undiagnosed and, subsequently, not receive adequate treatment for their influenza. The clinical importance of the early identification of influenza was underscored by our recent surveillance data. Out of the total suspected influenza samples processed using rRT-PCR method, 11 (11%) were positive for influenza virus. Subtyping of these 11 samples further showed that one sample and five samples were positive for influenza type A, subtypes H1 and H3, respectively, whereas five samples were positive for influenza type B virus.
A study was carried out at Chennai in the year 2002, in which out of 240 infants and children (0-12 years) with ARIs, 12.5% (30/240) were positive for influenza virus, of which 10% (24/240) were positive for influenza A (H3N2) virus and 1.66% (4/240) were positive for influenza B/Sichuan virus. Also, 0.833% (2/240) was positive for influenza type A (H3N2) virus and influenza type B/Sichuan virus strain. 
A similar study was carried out at Delhi during 2005-2007 among paediatric patients with lower respiratory tract infection, where 301 clinical samples were processed by multiplex PCR, and it was found that 3% (9/301) were positive for influenza A virus. 
Similar results were obtained in some studies with respect to influenza positivity in children, where influenza type A virus was found to be higher than influenza type B virus. , Analysis of the monthly data from 2007 and 2008 showed that influenza activity peaked during September-October as well as during the monsoon season. During the year 2009, influenza type A (H1) positivity was observed from April to July, prior to 2009 H1N1 influenza pandemic phase. In the present study, we observed no significant difference between influenza A and influenza B positivity during the study period.
Various surveillance studies have demonstrated that that relative humidity, rainfall and differences in temperatures influence the outbreaks of Influenza. In countries with temperate climate, influenza outbreaks occur in winter. ,,
In conclusion, our findings showed positivity of influenza virus in paediatric population in Mumbai region. Continuous monitoring would be required for early detection of any antigenic variants to understand the seasonality and analyse factors such as temperature, rainfall and humidity in the transmission of influenza viruses. Asymptomatic influenza accounts for approximately two-thirds of all influenza infections as determined by serological surveys. We emphasise on monitoring of influenza in different geographical locations, which will be helpful in paediatric patient management who are at risk for influenza complications. Also, healthy children with moderate to severe respiratory illness should be tested for influenza and, when infected, treated with oseltamivir or zanamivir.
| ~ Acknowledgments|| |
The authors thank Dr. MS Chadha, Assistant Director, and all the staff members in the Influenza Lab, National Institute of Virology, Pune, for technical support. The authors thank the WHO Collaborating Center for Influenza, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, for providing the reagents and necessary protocols, and the Dean, B. J. Wadia Hospital for Children, Mumbai, for the clinical samples. They are also grateful to Dr. S Muley for statistical analysis.
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[Figure 1], [Figure 2], [Figure 3]