Iranian Journal of Medical Microbiology

Study Design, Setting, and Participants’ Selection
This cross-sectional study was conducted from February 2022 to December 2023 in the paediatric ward and neonatal intensive care unit (NICU) in a North Indian Hospital (ESIC Medical College and Hospital, Faridabad). The study enrolled symptomatic children younger than five years who were admitted to the NICU or paediatric units after exhibiting the respiratory symptoms fulfilling the case definitions. In order to maintain the focus on respiratory diseases other than COVID-19, only SARS-CoV-2-negative cases were included.
A total of 927 respiratory samples were included. To ensure the sample integrity, respiratory specimens were collected in accordance with accepted clinical practices for the specimen processing. The NP/OP specimens were collected within 48 hours of starting the antibiotic treatment using sterile flexible flocked nylon swabs. The samples were stored at 4°C for up to 24 hours, and then transferred to -70°C (3).
Case Definitions
The ARI was defined as any illness with fever, cough, nasal congestion, shortness of breath, or sore throat, with an acute onset (within seven days). Severe acute respiratory infection (SARI) was defined as patients requiring overnight hospitalization due to cough that started within the past seven days. For infants under two months, SARI included any physician-diagnosed acute lower respiratory infection necessitating the overnight hospitalization.
Data Collection
Each participant's environmental, clinical, and demographic information was documented. Age, gender, low birth weight, breastfeeding habits, indoor air pollution exposure, malnourishment, vaccination status, presence of smokers at home, and vitamin A supplementation were important factors. Clinical signs including fever, abnormal white blood cell (WBC) counts, antibiotic response, and detected respiratory infections were also recorded. Interviews with caregivers and medical records were used to collect data.
The study protocol was approved by the Institutional Human Ethics Committee (Approval number 134 X/11/13/2022-IEC/01) and demographic and clinical details of the patients were collected after obtaining written informed consent from the guardian of the participants. GraphPad Prism version 8.0 was applied to perform the statistical analysis. To assess the relationships between categorical data, like particular infections and clinical or demographic outcomes, the chi-square test was used. A P-value less than 0.05 was defined as statistically significant correlation between the variables.
Nucleic acid extraction and Real-Time PCR
Nucleic acids were extracted using the QIAamp Viral RNA Mini Kit (Hilden, Germany). The TRUPCR® Respiratory pathogen panel kit was used to detect 33 respiratory pathogens. The bacterial pathogens detected using the respiratory panel were Klebsiella pneumonia, Mycoplasma pneumoniae, Streptococcus pneumonia, Staphylococcus aureus, Acinetobacter baumanii, Bordetella species, Chlamydia pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Pseudomonas aeruginosa, Legionella pneumophila, Haemophilus influenzae, and Moraxella catarrhalis. The viruses included parainfluenza 1-4, influenza A virus, (H3N2, Pandemic H1N1), influenzae B virus, human metapneumo virus (A+B), enterovirus, adenovirus, human respiratory syncytial virus, human rhinovirus, and human bocavirus.
Fluorophore-labelled probes specific for various targets were used. The amplification conditions consisted of cDNA synthesis followed by 40 cycles of PCR. Standard RT-qPCR reactions were performed with 20 μl of RT-qPCR mix containing master mix, primer/probe mix, enzyme, and extracted nucleic acid. The positive and negative controls were used to verify the reaction efficiency and to detect any contamination, respectively.

Figure 1. Distribution of mono-infections and co-infections among the ARI cases.

Figure 2. Temporal distribution of respiratory viruses during the study.


Figure 3. Month-wise positivity trend of respiratory viruses during pandemic and post- pandemic with respect to SARS-CoV-2 virus (4).


Figure 4. Month-wise positivity trend of respiratory viruses before and after SARS-CoV2 pandemic (5, 6).

The study highlights key findings regarding acute respiratory infections (ARI) in paediatric populations. Out of total cases, 386 ARI cases originated from the paediatric wards, while 43 cases were from intensive care units (ICUs), predominantly affecting infants, particularly males. This observation aligns with previous research by McClelland et al (7), Juliana et al (8), and Zhao et al (9) suggesting gender-specific immune response differences may contribute to this trend. Real-time PCR analysis revealed 34 cases with co-infection and 395 cases with mono-infection, emphasizing the complexity of ARI cases and the potential for multiple pathogen involvement simultaneously, consistent with existing literature (10-12).
The seasonality of respiratory viruses, such as influenza, human coronaviruses, and RSV demonstrates winter peak episodes, while adenovirus, human bocavirus, rhinovirus, and hMPV are present year-round. According to studies by Peci et al (13), Midgley et al (14), Killerby et al (15), Monto et al (16), Landes et al (17), Morikawa et al (18), Bastien et al (19), Haynes et al (20), Abedi et al (21), Lee et al (22), and Monto (23), the enteroviruses, known as "summer viruses," exhibit increased counts during warmer months, while rhinovirus severity peaks in winter. Although the findings of the current study on RSV align with previous studies, the higher incidence of human metapneumovirus could be due to variations in the respiratory viruses’ epidemiology, variations in the study populations, or advancements in the diagnostic techniques as suggested by Falsey et al (24), Atmar et al (25), Li et al (26), and Suminda et al (27).
The COVID-19 pandemic altered the seasonality of influenza and RSV infections globally, with off-season spikes following declines during COVID-19 peaks (28-30).
Several theories attempt to explain the decrease in influenza and RSV cases during SARS-CoV-2 peaks, including viral antagonism and NPIs. Post-pandemic influenza subtypes have shifted with changes in circulation patterns observed (31).
Studies in India during the pandemic and post-pandemic era highlight shifts in the respiratory pathogens circulation, with varying prevalence and seasonality observed (4-6). Our study reflects these trends, with clear seasonal patterns in respiratory viruses’ prevalence, particularly during colder months. RSV, PIV, and adenovirus peak towards the end of the year, while influenza A H3N2 peaks in November and influenza B in October. Rhinovirus and hMPV exhibit intermittent activity throughout the year, with sporadic peaks. Overall, our findings contribute to understanding the dynamic nature of respiratory viruses’ circulation and the impact of the COVID-19 pandemic on seasonal patterns and prevalence.

 

The study limitations include its restricted generalizability to other age groups or geographical areas due to the small sample size. Recall bias and the use of self-reported information and medical records may result in potential errors. Furthermore, knowledge of infection dynamics and the discovery of possible biomarkers are impeded by the lack of immunological analysis and the limited investigation of long-term health repercussions, which ultimately impacts the formulation of the successful treatment plans.
The study found a strong association between the acute malnutrition and early ARIs, supporting previous research that identifies malnutrition as a key contributor to the childhood ARIs. This link may stem from malnutrition role in compromising immune function, increasing vulnerability to the infections (32, 33).
Additionally, the detrimental effects of inadequate breastfeeding on child health highlight the need for the targeted health promotion campaigns. Such initiatives should focus on providing support for the breastfeeding through community health workers, lactation consultants, and support groups.

 

The study underscores significant associations between various demographic factors and ARI prevalence, including age, gender, low birth weight, breastfeeding practices, household conditions, exposure to indoor air pollution, and malnutrition. These findings highlight the multifactorial nature of the ARI and emphasize the importance of addressing socio-environmental determinants in disease prevention and management. By examining the viral evolution dynamics and its correlation with respiratory infections epidemiology in children, the study aimed to anticipate and address the emerging threats to paediatric respiratory health in the post-pandemic period. Given the on-going repercussions of the COVID-19 pandemic, it is crucial to comprehensively examine the risk factors for the ARIs in children. This research contributes to understanding the multifaceted aspects of the social interaction, immune system alterations, and viral evolution, aiming to inform targeted interventions and public health strategies to safeguard children's respiratory health in the post-pandemic era. Understanding the impact of post-pandemic respiratory infections on children requires a thorough exploration of the various involved risk factors. As we delve deeper into these dynamics, it becomes increasingly clear that a holistic approach is essential for developing effective interventions and public health strategies.

 

The authors would like to acknowledge the study participants for their cooperation.
 
 

Ethical Considerations

The study protocol was approved by the Institutional Human Ethics Committee (Approval number 134 X/11/13/2022-IEC/01). Demographic and clinical details of the patients were collected from the guardians of the participants after obtaining written informed consent.

Authors’ Contributions

Aparna Pandey conceptualized, supervised, and administered the study. Arundhati Biswas and Priti Agarwal conducted the formal analysis. Pooja Pandey, Aparna Pandey, Anil K Pandey, Rajiv M Gupta and Asim Das conducted the investigation. Priti Agarwal, Arundhati Biswas and Pooja Pandey wrote the main manuscript text and edited. All authors reviewed and approved the manuscript.

There is no grant support or financial relationship associated with this study
 
 

Conflicts of Interest