Evaluation of Respiratory Virus Pathogen from Patients Presenting with Influenza like Illness at Various Hospitals in Delhi, India

Background: Respiratory viruses have been widely circulating in human populations and its variants have caused, and continue to cause, substantial morbidity and mortality worldwide. Objective:The objective of this study was to compare the presence of various circulating respiratory virus among the population in Delhi region. Materials and Method: The epidemiology and burden of human respiratory viruses were examined in a cohort of 106 patients from Delhi, India, by using real-time quantitative reverse transcription polymerase chain reaction. Results: Of the 106 screened samples tested,35.84% patients were found positive for influenza A virus, 19.81% for Human Metapneumovirus (hMPV),5.66% for Rhinovirus (HRV), 3.77%for Parainfluenza (PIV) type 4 and Human Enterovirus (HEV), 2.83% for Human Coronavirus (HCoV) OC43, while 0.94% for influenza B, coronavirus NL63 and 229E, Parainfluenza type-2 and 3 and Bocavirus. The analysis revealed that Parainfluenza (PIV-1, 2 and 3), Respiratory Syncytial Virus (RSV) A and B, and Human Bocavirus (HBoV) are not commonly circulating among general population. Conclusion: The metapneumovirus and influenza A virus are important respiratory pathogens in patients. Apart from these HRV and HEV embark to the clinical significance in child care. Influenza and HRV caused the highest-impact illnesses. The current study depicts the burden of respiratory viruses in current population.


Introduction
Acute Respiratory Infections (ARI) are a major global public health problem and widely circulating in human population. Despite progress made in the 20 th century with the introduction of antibiotics, vaccines and antivirals, there are no specific interventions for most respiratory infections of viral origin. Although, the recent years have witnessed a tremendous development in the management of respiratory viruses including influenza and other respiratory viral infections, the world still suffers from these viral infections every year leading to an increase in morbidity and mortality [1][2][3][4].
These infections continue to cause frequent morbidity, and sometimes cause severe outcomes including death, especially in developing countries [5][6]. ARI can be the leading cause of morbidity in young children, and accounted for one-fifth of all deaths in children less than five years of age. Of those mortalities, 70% occurs in Africa and Southeast Asia [7]. Approximately one-third of children develop Lower Respiratory Tract Infections (LRTI) in the first year of life [8].
The major causes of Acute Respiratory Distress Syndrome (ARDS) in children and adults are influenza A and B viruses, Parainfluenza Virus (PIV) type 1-3, Respiratory Syncytial Virus (RSV), adenovirus, and rhinovirus. Other viruses such as Coronavirus (CoV), human bocavirus, human enterovirus, PIV-4, the newly discovered parvovirus types 4 and 5, and mimivirus also infect the respiratory tract albeit at a much lower frequency [9][10][11]. Rhinoviruses and CoVs were identified as human pathogens in the 1960s [12], but they have been largely ignored by the medical community because their clinical impact was considered to be minor. The rhinoviruses and CoVs, once thought to cause only common cold, can cause LRTI and ARDS and can be fatal in few cases. Indeed, all of the viruses mentioned above have overlapping clinical presentations and cause both upper respiratory tract infection (URTI) and LRTI, and cannot be distinguished by attending physicians unless a laboratory diagnosis is done for the causative agent [11].
Influenza virus is known to cause annual epidemics in temperate climates that are characterized by a sudden increase in febrile respiratory illness; the epidemic period is generally 3 to 8 weeks [11,12]. Although the four serotypes of PIV that infect human has the similar mode of transmission and pathogenesis, they don't undergo antigenic shift and drift [13]. PIV-1 is the major cause of acute croup in infants and young children but also causes mild URTI, pharyngitis, and tracheobronchitis in all age groups. PIV-2 is generally associated with lower infection rates than PIV-1 or PIV-3 and has been associated with mild URTI, croup in children, and, occasionally, LRTI. PIV-3 is a major cause of severe LRTI in infants and young children, often causing croup, bronchitis, and pneumonia in children <1 year of age [11]. In older children and adults, it can cause URTI or tracheobronchitis [14].
RSV is another etiological agent causing respiratory disease in infancy and is a major cause of bronchiolitis and pneumonia in infants under 2 years of age [15]. hMPV causes both URTI and LRTI with similar symptoms ranging from mild rhinorrhea associated with common colds to severe cough, wheezing, bronchiolitis, and pneumonia [15,16].The adenovirus is pervasive, and infections are prevalent; only the serotypes 1 to 5, 7, 14, 19, and 37 infect the respiratory tract, causing a variety of mild symptoms including fever, rhinitis, pharyngitis, cough, and conjunctivitis and more severe disease including laryngitis, croup, bronchiolitis, or pneumonia, on occasion [17,18]. Most adenovirus infections occur early in life, and by age of 10 years, most children are infected with at least one serotype [19].
Human rhinoviruses are perhaps the most captivating of the conventional viruses as revealed by the recent reports of higher-than-expected infection rates in hospitalized children with acute LRTI [20-25]. Rhinoviruses were believed to cause only "the common cold" and thus were neglected by the medical community until not long ago, when their spectrum of disease expanded.
Among other respiratory viruses, the enteroviruses are ubiquitous agents found worldwide [26]. It has been estimated that enteroviruses infect 1 billion or more individuals worldwide each year. Several modes of transmission exist for these viruses, including fecal-oral, respiratory, transplacental, perinatal, and self-inoculation modes, but the majority are fecal-oral [27,28]. The highest incidence of enterovirus infection is in infants and young children [29]. The majority of enterovirus infections are asymptomatic [30]. HCoV-OC43 and HCoV-229E were identified in the mid-1960s as being a cause of mild self-limited URTI and were subsequently shown to cause about onethird of "common cold"-like illnesses in adults. Like other respiratory viruses, HCoV-OC43 and 229E are spread by large droplet infection. Overall, they account for 5 to 30% of respiratory tract infections, and outbreaks may occur in 3 to 4 years interval [31]. HCoV-OC43 and 229E has been associated with both URTI and LRTI in a variety of settings including nosocomial infections in high-risk immune-compromised children, in hospitalized elderly patients with non-influenza-virus ARD and pneumonia, and in newborns, children and hospital staff [32][33][34]. The recent findings by the infection control practitioners show that HCoV-229E can survive for up to 3 hours when dried on solid surfaces and for up to 6 days in saline solution at room temperature [35].
In most studies where the frequencies of several viruses were determined, HBoV was most prevalent in children <3 years of age and less common than RSV and rhinovirus. HBoV infections occur in both children and adults, but children under the age of 2 years appear to be mostly at risk of infection [35].

Study design
We retrospectively studied all critically ill patients with Influenza Like Illness (ILI) from Military Base Hospital, Delhi; Kalawati Saran children Hospital, Delhi and V.P. Chest Institute, Delhi from September, 2012 to August, 2013. The patients with influenza-like-illness and respiratory complications presenting to the Out-Patient Department (OPD) during the study period underwent diagnostic testing for evidence of a panel of respiratory virus [36].Appropriate clinical specimens (throat swabs and nasal swabs) were collected in viral transport medium, as described previously following Centers for Disease Control and Prevention (CDC), Atlanta and WHO guidelines prior to informed consent from the patients or guardian [37][38][39]. All the collected clinical specimens were subjected to one-step Reverse Transcriptase-PCR (RT-PCR) for a panel of respiratory viruses, from Seeplex RV15 one-step Ace detection kit (Seegene, Korea) as per manufacturer's instructions. Ethical clearance for conducting the study was obtained from the Institute's Ethics Committee.

Statistical analysis
Continuous variables were presented as the mean ± standard deviation (SD) and median with interquartile ranges. Categorical variables were presented as numbers and percentages. The Statistical graphs were made through analyzing data with Graph Pad Prism software version 5.5.

Result
During the stipulated study period, patients with respiratory illness were evaluated; a total of 106 patients of different age group were screened.
The mean age of patients infected with influenza was 33.55+19.16followed by 9.69+13.65 for hMPV, 3.49+3.80 for HRV, 25.72+40.95 for PIV4, 0.41+ 0.58 for coronavirus OC43, 4.93+3.71 for HEV and single cases of influenza B, coronavirus NL-63 and 229E, HBoV, PIV type 2and3. In comparison, the mean age was found to be 3.83+3.48 for patient group below 18 years, followed by 43.23+16.93 for patient group above 18 years. It was observed that the most of the cases were in the range of 0-18 year age group (Figures 1 and 2).
Although, the climatic conditions during the study period are same for both the study groups but it was found that the patients below age of 18 year shows maximum severity in terms of symptoms ( Figure  3).The rate of respiratory infections were also more frequent in below 18 year age group (Figures 4.1 and 4.2). Also, the study reveals that the extensive pathogen infecting patients above 18 years, age group is Influenza A (71.6%) while it is Metapneumovirus (39.02%) for patients below 18 age group ( Figure  2).

Discussion
The viruses continue to pose novel challenges by donning new disguises that manage to outwit our immune system. The data on the clinical presentations of respiratory viruses needs to be extensively studied. This surveillance data confirms the virulence and strain circulatory patterns in different age groups in Delhi.
Although both the age groups were reported to have respiratory viral infection, however, our findings show that the group ranging 0-18 years was found to be infected more frequently as compared to other older groups (Figures 4.1and 4.2). The result depicts that only the patients below 18 years, was more prone to infection by different respiratory viral flora rather than the other patient group (>18years). The young age which might acts as a substrate factor reflecting the reciprocity of factors causing disease following viral infection. The severity of infection can be contributed to the age of child which has an effect in terms of the airway size, transmission dynamics, and immune experience of the child. As younger children have smaller energy reserves, they tends to get exhausted by the effort during breathing, which can be concluded for mortality in acute bronchiolitis. There are also critical differences in the infant immune system compared to that of adults that directly affect infection [40]. ILI can be attributed to a wide range of respiratory viruses, including influenza viruses, adenoviruses, RSV, HEV, HRV, hMPV, HBoV, CoV, and PIV. Patients infected by these diverse viral pathogens develop similar symptoms which transcribe unreliable clinical diagnosis and limit etio-epidemiological studies [41].
The proportion of infectivity for both influenza virus and metapneumovirus were quite distinguishable and reveals that, the both virus caused maximum illness, in all age group as it includes higher rates of reported cough, breathlessness and bodyache to the patients ( Figure  3) .The higher percentage of symptomatic cough in viral infection may indicate substantial cellular damage to the respiratory epithelium. The first days of a viral URTI are often associated with a dry, unproductive cough, which may be caused by the inflammatory response in the upper airways spreading to the larynx and cause loss of sleep and exhaustion. Cough associated with URTIs is believed to be caused by a hyper-reactivity of the cough reflex that may be due to the effects of inflammatory mediators on airway sensory nerve endings [42]. Cough occurs spontaneously with an URTI, and some cough may be voluntary rather than reflex; this voluntary cough may be related to a sensation of airway irritation [43]. Productive cough usually occurs later in the course of URTI and may be related to the inflammation spreading to the lower airways and triggering mucus production.
The high rates of breathlessness and bodyache is contributed to the formation of free radical and proinflammatory cytokines which ultimately cause oxidative stress during viral infection. In context to free radical formed in airway nitric oxide (NO) is merely important and is almost unreactive radical except for its termination reaction with superoxide radical to yield the strong oxidant peroxynitrite. Excess of NO species assumed to be directly related to hypotension and septic    shock and endotoxemia to produce muscle dysfunction and oxidative stress. Myalgia is a common symptom of URTIs, contributing 50% of common cold in patients [44,45]. Fever associated with URTIs is usually accompanied by other systemic symptoms such as myalgia, indicates that both these symptoms are caused by the production of prostaglandin E2 in response to circulating cytokines [46,47].
These findings show the extent to which a respiratory virus put burden among the different age. Early diagnosis improves the efficiency of prophylaxis or treatment by antivirals and has a strong impact on the cost-effectiveness of curative treatment. A microbiological diagnosis may have a significant impact on decreasing excessive and inappropriate use of antibiotics [48]. Excessive antibiotic use is being increasingly recognized as the main selective pressure driving resistance. In a study investigating outpatient antibiotic use in 26 countries in Europe, there was a shift from the old narrow-spectrum antibiotics to the new broad-spectrum antibiotics and there were higher rates of antibiotic resistance in higher consuming countries [49]. One of the consequences of over-prescribing antibiotics, especially to individuals with viral infections only, is that no clinical improvement is observed and the potential risk to cause antibiotic resistance increases.
As new antiviral options become available, better treatment choices will benefit the patient and community. Molecular techniques provide timely information regarding the identification of pathogens detected and will guide the clinician's choice of therapy [50].
The observations documented in the present study provide an insight to the epidemiology and clinical manifestation of respiratory viruses in the common population in northern India and may help clinicians in making an early diagnosis, help to understand the roles that pathogens play in particular infection so as to institute appropriate treatment and management. Also, the counseling should be instituted through clinicians or health care workers to the patients and their family.