Leveraging real-time PCR for early diagnosis of respiratory conditions in the Indian pediatric population

INTRODUCTION

Respiratory viruses and microbial pathogens are widely distributed and result in a diverse range of clinical symptoms. Respiratory tract infections (RTIs) are highly prevalent, and identifying the causative pathogen is crucial for proper management.¹ Besides well-known respiratory viruses like respiratory syncytial virus (RSV) and influenza virus, human metapneumovirus (MPV) was discovered in 2001, followed by the identification of other respiratory viruses.^2,3 The disease burden of respiratory viruses is currently not fully understood. These viruses have been found in over two-thirds of children with radiographically confirmed community-acquired pneumonia (CAP). Similarly, in the United States, molecular diagnostics revealed viral infection in 43%-67% of pediatric CAP cases.² Respiratory viruses also play a significant role in adult pneumonia, being detected in 15%-56% of adult CAP cases.^2,4 They are responsible for the majority of respiratory infectious diseases in both children and adults, leading to a substantial disease burden.^5,6 Additionally, identifying the causative viruses allows for accurate diagnosis of respiratory infections, prescription of specific antiviral agents against certain viruses (e.g., oseltamivir for influenza viruses), and improves prognostic evaluation Recognizing causative viruses can also inform appropriate infection control measures, potentially reducing unnecessary hospital stays and enabling the discontinuation of unnecessary antibiotics.^2,7-9 In summary, respiratory virus infections are common, and testing for respiratory pathogens can enhance understanding of their roles in respiratory diseases and contribute to improved clinical management.¹⁰

The timely and accurate diagnosis of viral infections can be challenging. Rapid antigen tests are used worldwide to detect influenza virus infection, but concerns exist regarding the sensitivity of currently available viral antigen tests.¹¹ Technological advancements have improved the sensitivity, accessibility, and utility of viral diagnostic tools.^12,13 Molecular assays have been developed and progressively multiplexed to diagnose numerous respiratory viruses in a single assay with excellent sensitivity and specificity.¹⁴ The importance of molecular-based diagnostic modalities is increasing, with polymerase chain reaction (PCR) technology being increasingly utilized in clinics to rapidly diagnose respiratory infections¹⁵. This study aims to detect respiratory viruses in children using PCR and to compare the detection power of this technique against that of traditional antigen tests. The clinical conditions were also investigated.

METHODS

Study type

This study employed a descriptive approach to analyse respiratory pathogens in children presenting with respiratory symptoms suspected of viral or bacterial infections.

Study place

The study was conducted at Lifecell International Pvt Ltd., a clinical laboratory located in Chennai, India.

Study period

This study was conducted from August 2023 to February 2024.

Sampling technique/selection criteria of patients

Non-probability sampling: Children with respiratory symptoms and clinical suspicion of viral or bacterial infections are referred to Lifecell International Pvt Ltd. by pediatricians for routine diagnostic testing. This method suggests that participants are selected based on their availability and the clinical judgment of referring pediatricians, rather than through random selection from a larger population.

Procedure

Nasopharyngeal swabs were collected from each participant by pediatricians using sterile swabs and placed in 2.5 ml viral transport medium. Specimens were processed to extract nucleic acids using the Applied Biosystems MagMAX Viral/Pathogen II Nucleic Acid Extraction Kit and the Kingfisher Flex automation instrument.

Nucleic acid extraction and real-time PCR

Nucleic acids were extracted from nasopharyngeal swabs using the Applied Biosystems MagMAX Viral/Pathogen II Nucleic Acid Extraction Kit and the Kingfisher Flex automation instrument. Real-time PCR, based on TaqMan technology, was employed to detect 32 respiratory pathogens, including:

Bacteria: Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Mycoplasma pneumoniae, Salmonella spp., Streptococcus pyogenes, Bordetella spp., Chlamydia pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Legionella pneumophila, Haemophilus influenzae (AF), Moraxella catarrhalis.

Viruses: Human parechovirus, SARS-CoV-2, Human parainfluenza virus 1-4, Influenza A virus, Pandemic H1N1 influenza virus (PDM H1N1), Influenza A (H3N2) virus, Influenza B virus, Influenza C virus, Enterovirus, Human metapneumoviruses (A/B), Human adenovirus, Human respiratory syncytial viruses (A/B), Human rhinovirus, Human bocavirus (Table 1).

Ethical approval

This study was approved by the Institutional Review Board of Lifecell International Pvt Ltd., Chennai.

RESULTS

The study included a total of 100 paediatric participants, comprising 70 males and 30 females, distributed across various age groups (Table 2). Table 3 presents the age-wise distribution of participants.

The majority of participants were in the age group of 1 month to 12 months (38%), followed by those between 1 and 3 years (29%), and above 3 to 10 years (25%). Infants below 1 month of age constituted the smallest proportion (8%) of the study population (Table 3).

As multiple pathogens were identified in some samples, the total count of pathogens reached 188 despites only 100 samples being studied.

Among males, the highest number of positive cases was observed in the age group below 1 month, with 7 out of 7 cases (100%), followed by the age group between 1 month to 12 months, where 28 out of 29 total cases were positive (96%). Similarly, the age group between 1 and 3 years had 100% positivity with 18 positive cases out of 18 total cases. In older age groups, all males above 3-10 years of age tested positive (100%). For females, all age groups studied showed a 100% positive rate except for the 1-12 months age group, which had a 96% positive rate. The distribution of positive cases for a specific condition among different age groups and genders is detailed in Table 3.

In total, 134 pathogens were identified across 100 patients, exhibiting a predominant occurrence in males (80%) relative to females (20%). Among the diverse microbial entities identified, Human respiratory syncytial viruses A and B emerged as the most prevalent, constituting 17 cases in males and 5 in females. Following closely, Klebsiella pneumoniae ranked as the second most prevalent pathogen, with 19 cases detected in males and 5 in females. Noteworthy prevalence was also observed for Influenza A virus and Influenza B virus, with 7 and 9 cases respectively in males, and 4 and 7 cases in females. Additional notable pathogens included Human parainfluenza virus 3, Human adenovirus, and Streptococcus pneumoniae, each demonstrating considerable occurrence across both genders. It is noteworthy that certain pathogens, such as SARS-CoV-2 and Acinetobacter baumannii, were absent in either gender throughout the observation period. These findings underscore the diversity of microbial infections and underscore the significance of gender-sensitive analysis in elucidating their epidemiology and implications for public health. Table 4 presents a comprehensive summary of microbial infections discerned among male and female individuals.

Male samples exhibited a higher rate of co-infection compared to female samples.

Statistical analysis reveals that out of the total 38 cases, 29 (76.3%) were in males and 9 (23.7%) were in females. Among the various coinfection combinations, the most prevalent was Human respiratory syncytial viruses A and B coexisting with Klebsiella pneumoniae, with 7 cases recorded. This was followed by coinfections involving Human parainfluenza virus 3 and Haemophilus influenzae (A-F), as well as Human metapneumo viruses A & B and Haemophilus influenzae (A-F), each with 5 cases. Notably, coinfections involving Influenza B virus and Haemophilus influenzae (A-F) exhibited gender disparity, with 3 cases in males and 1 case in females. Additionally, coinfections involving Enterovirus and Klebsiella pneumoniae showed equal distribution between males and females, with 1 case each. These findings underscore the complexity and diversity of coinfections, highlighting the need for further research to understand their clinical implications and management strategies. The table 5 provides a detailed breakdown of coinfections involving more than two pathogens detected in individuals, categorized by pathogen combinations and gender.

DISCUSSION

In contemporary clinical environments, the swift and precise diagnosis of respiratory viruses has become paramount. Not only does this aid infection control teams in hospitals to mitigate the transmission of highly virulent or resistant pathogens, but it also ensures timely interventions for vulnerable populations such as premature infants and individuals with underlying health conditions.

Our study demonstrates that PCR significantly outperforms traditional diagnosis. Utilizing PCR-based viral detection allows physicians to make more informed decisions, reducing unnecessary antibiotic or antiviral usage. Accurate identification of specific viruses enables prompt antiviral treatments, such as oseltamivir for RSV infections, and appropriate antibiotic therapies, like cefotaxime for Haemophilus influenzae. Utilizing TaqMan probe assays covering a panel of 32 pathogens, we identified 188 microbial pathogens, including viruses, bacteria, and atypical bacteria. This comprehensive approach facilitated enhanced pathogen detection

Our research found RSV and Haemophilus influenzae to be the most prevalent pathogens among pediatric patients, especially in neonates and children aged 6 days to 3 years, with higher incidences in males (24.29% for RSV and 31.43% for Haemophilus influenzae) compared to females (16.7% for RSV and 10% for Haemophilus influenzae). Rapid diagnosis and early treatment of RSV with palivizumab or ribavirin, and Haemophilus influenzae with antibiotics, are essential. Timely identification also aids in implementing effective infection control measures.

Pathogen distribution varies by age, with children generally experiencing higher rates of viral infections than adults. Research indicates that in hospitalized adults with community-acquired pneumonia (CAP), rhinovirus (9%) and influenza viruses (6%) are predominant, while in hospitalized CAP children, respiratory syncytial virus (28%), rhinovirus (27%), and metapneumovirus (13%) are most common.

A study conducted by Yu Lin et al in 2020 investigated both symptomatic and asymptomatic premature infants in a neonatal ICU through prospective screening using multiplex PCR twice weekly. This screening identified respiratory viruses in 52% of the infants during their hospitalization. Infected infants experienced significantly longer hospital stays (70 days versus 35 days) and a higher incidence of bronchopulmonary diseases.²

Over the past decade, the substantial impact of respiratory viral infections on adult and pediatric patients, particularly those with hematologic malignancies, hematopoietic stem cell transplants, and solid organ transplants, has gained recognition. In these highly immunocompromised groups, respiratory viruses frequently progress to pneumonia, with mortality rates ranging from 30% to 50%. Several studies have demonstrated the utility of multiplex PCR in detecting respiratory viruses in these high-risk populations.^16-19

In our investigation, respiratory syncytial virus (RSV) was the most commonly detected pathogen in both males and females, followed by adenovirus and influenza B virus. These findings align with prior research, such as a study in Spain where RSV was the predominant pathogen. Our research further emphasizes the crucial role of PCR in identifying respiratory viruses, particularly in vulnerable populations like premature infants and immunocompromised patients. In the neonatal ICU, PCR screening revealed viral infections in more than half of prematurely born infants, leading to prolonged hospital stays and increased incidences of bronchopulmonary diseases.²⁰

Respiratory viral infections, often presenting with fever and respiratory symptoms, are prevalent and can be challenging to differentiate from bacterial infections, resulting in unnecessary antibiotic use. This misuse exacerbates the global challenge of antimicrobial resistance (AMR), necessitating accurate and prompt diagnosis to mitigate the risks associated with inappropriate antibiotic use.^{7, 21-26}

Our study underscores the effectiveness of nucleic acid-based tests, especially multiplex PCR, in detecting respiratory viruses in pediatric patients with respiratory tract infections (RTIs). A retrospective analysis revealed a male predominance across various age groups, highlighting the significant impact of respiratory viral infections in this demographic.

Expanded pathogen coverage led to a significantly higher rate of positive test results, with pathogens like Haemophilus influenzae (A-F), RSV, adenovirus, and Klebsiella pneumoniae frequently identified across all age groups. Male patients exhibited a higher prevalence of infections, with distinct pathogen profiles observed across different age categories. Additionally, coinfections involving multiple microbial pathogens were more prevalent in males, particularly in the 0-10 year’s age group, underscoring the importance of considering coinfections in clinical management.

India is among the 15 countries with the highest burden of pneumonia cases and related childhood mortality. Approximately 400,000 children under the age of five die each year in India due to acute respiratory infection (ARI) diseases, accounting for 13-16% of all child deaths among pediatric hospital admissions.^27,28 In children, exposure to Haemophilus influenzae, particularly type b infections, poses a risk of secondary infection, with approximately a 2.1% likelihood among contacts. This highlights the importance of vigilance and preventive measures, as adults in close contact with infected children must be aware of the potential for transmission. Preventive strategies such as vaccination are crucial in reducing the spread of H. influenzae infections and protecting vulnerable populations, including children, from severe outcomes associated with this pathogen.²⁹

A study of children aged 4-5 years reported a 47.3% prevalence rate of ARI, while we observed a higher prevalence among infants. Community-based studies in coastal Karnataka and urban West Tripura, India, also reported a higher incidence of pneumonia among infants.^30-32 ARI prevalence was highest among infants aged 0-12 months (63.2%), followed by children aged 25-60 months (59.5%), and was lower in those aged 13-24 months (52.6%). Boys (62.9%) had a higher prevalence of ARI than girls (55.1%). Higher ARI rates were also observed in children with birth weights under 2.5 kg, those whose mothers had education levels between 1st and 7th grade, those with two or more siblings, and those living in overcrowded conditions.³³

A comparison between rural Kenya and North America highlights differing healthcare challenges. In rural Kenya, infant and child mortality rates remain high at 60 and 20 per 1000 live births, respectively, despite a nearly 50% reduction in infant mortality from 2003 to 2010, largely due to decreased deaths from respiratory infections. By 2010, malaria had surpassed respiratory infections as the leading cause of infant mortality. In contrast, a U.S. study reported hospitalization rates for radiologically proven pneumonia at 6.2 per 1000 infants and 1.6 per 1000 children, with only three deaths among 2358 admitted children. Notably, 33% of these children were diagnosed with asthma or reactive airways disease. The low mortality rates in developed countries are likely due to factors such as vaccination, access to antibiotics, good nutrition, low biofuel exposure, and improved sanitation.^34-37

In the study by Jain et al bacterial pathogens were identified in a minority of cases, with viruses being more common. Respiratory syncytial virus (RSV) was prevalent in younger children, while rhinovirus was more common in older children. In 15-30% of cases, more than one virus or a combination of virus and bacteria were identified, varying by age. While identifying a "respiratory pathogen" in acutely ill subjects might suggest a causal relationship, it is known that bacteria like Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae are often found in asymptomatic individuals. Similarly, some studies show that certain respiratory viruses are more frequently detected in asymptomatic children than in those with symptoms.³⁵

Principi et al conducted weekly upper airway sampling in 88 infants, finding that 64% of the 326 rhinovirus-positive samples were from asymptomatic infants. Symptom likelihood was not significantly affected by rhinovirus type (A, B, or C), and no infants were hospitalized.³⁸ Chonmaitree et al identified viruses in 39% of samples, with rhinovirus being most common; 28% of asymptomatic infants had viruses, accounting for 53% of positive samples. The odds of viral identification in symptomatic subjects were 11.7 times higher than in asymptomatic infants with RSV.³⁹ Rhedin et al found RSV, hMPV, influenza, and adenovirus were more common in community-acquired pneumonia than in healthy controls, unlike rhinovirus and bocavirus.⁴⁰ Shi et al.'s systematic review confirmed these findings, showing strong causal links for RSV (OR 9.8), influenza (OR 5.0), parainfluenza (OR 3.4), and hMPV (OR 3.8).⁴¹

Though the study underscores the necessity of molecular diagnosis for pediatric respiratory illnesses, it has several limitations. The small sample size of 100 pediatric participants restricts the generalizability of the findings. Additionally, the use of non-probability sampling and reliance on clinical suspicion for participant inclusion may introduce selection bias, impacting the representativeness of the study population. To enhance the validity and sensitivity of the findings, further research with a larger sample size is required.

CONCLUSION

In conclusion, our study highlights the evolving landscape of respiratory virus detection and emphasizes the importance of PCR-based methods in improving patient outcomes and guiding infection control measures. Continued research efforts are necessary to elucidate the clinical significance of viral coinfections and optimize diagnostic and management strategies for respiratory tract infections in pediatric populations.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the Institutional Review Board of Lifecell International Pvt Ltd., Chennai (LC-ET-2411)

REFERENCES

1. 1. Mills JM, Harper J, Broomfield D, Templeton KE. Rapid testing for respiratory syncytial virus in a paediatric emergency department: benefits for infection control and bed management. J Hosp Infect. 2011;77(3):248-51.
2. 2. Lin CY, Hwang D, Chiu NC, Weng LC, Liu HF, Mu JJ, et al. Increased detection of viruses in children with respiratory tract infection using PCR. Int J Environm Res Publ Heal. 2020;17(2):564.
3. 3. Crowe JE, Williams JV. Paramyxoviruses: respiratory syncytial virus and human metapneumovirus. Viral Infect Humans: Epidemiol Control. 2014:601-27.
4. 4. Pavia AT. What is the role of respiratory viruses in community-acquired pneumonia?: What is the best therapy for influenza and other viral causes of community-acquired pneumonia?. Infect Dis Clin. 201327(1):157-75.
5. 5. Avendaño Carvajal L, Perret Pérez C. Epidemiology of respiratory infections. Pediatric respiratory diseases: a comprehensive textbook. 2020:263-72.
6. 6. Shapiro ED. Epidemiology of acute respiratory infections. InSeminars in Pediatric Infectious Diseases. WB Saunders. 1998;9(1):31-6.
7. 7. Breakell R, Thorndyke B, Clennett J, Harkensee C. Reducing unnecessary chest X-rays, antibiotics and bronchodilators through implementation of the NICE bronchiolitis guideline. Euro J Pediatr. 2018;177:47-51.
8. 8. Jain S, Williams DJ, Arnold SR, Ampofo K, Bramley AM, Reed C, et al. Community-acquired pneumonia requiring hospitalization among US children. New Eng J Medi. 2015;372(9):835-45.
9. 9. Jain S, Self WH, Wunderink RG, Fakhran S, Balk R, Bramley AM, et al. Community-acquired pneumonia requiring hospitalization among US adults. New Engl J Medi. 2015;373(5):415-27.
10. 10. Chen H, Weng H, Lin M, He P, Li Y, Xie Q, et al. The clinical significance of FilmArray respiratory panel in diagnosing community‐acquired pneumonia. BioMed Res Inter. 2017;2017(1):7320859.
11. 11. Bruning AH, Leeflang MM, Vos JM, Spijker R, de Jong MD, Wolthers KC, et al. Rapid tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review and meta-analysis. Clin Infect Dis. 2017;65(6):1026-32.
12. 12. Burbelo PD, Iadarola MJ, Chaturvedi A. Emerging technologies for the detection of viral infections. Future Virol. 2019;14(1):39-49.
13. 13. Sung CC, Chi H, Chiu NC, Huang DT, Sung CC, et al. Viral etiology of acute lower respiratory tract infections in hospitalized young children in Northern Taiwan. J Microbiol Immunol Infect. 2011;44(3):184-90.
14. 14. Zhang S, Zhang W, Tang YW. Molecular diagnosis of viral respiratory infections. Curr Infect Dis Repo. 2011;13:149-58.
15. 15. Yang S, Rothman RE. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis. 2004;4(6):337-48.
16. 16. Lee JH, Jang JH, Lee SH, Kim YJ, Yoo KH, Sung KW, et al. Respiratory viral infections during the first 28 days after transplantation in pediatric hematopoietic stem cell transplant recipients. Clini Transplant. 2012;26(5):736-40.
17. 17. Bennett NJ, Tabarani CM, Bartholoma NM, Wang D, Huang D, Riddell SW, et al. Unrecognized viral respiratory tract infections in premature infants during their birth hospitalization: a prospective surveillance study in two neonatal intensive care units. J Pediatr. 2012;161(5):814-8.
18. 18. Huang HS, Tsai CL, Chang J, Hsu TC, Lin S, Lee CC. Multiplex PCR system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis. Clin Microbiol Infect. 2018;24(10):1055-63.
19. 19. Sim SA, Leung VK, Ritchie D, Slavin MA, Sullivan SG, Teh BW. Viral respiratory tract infections in allogeneic hematopoietic stem cell transplantation recipients in the era of molecular testing. Biol Blo Marr Transplant. 2018;24(7):1490-6.
20. 20. Calderaro A, Buttrini M, Farina B, Montecchini S, De Conto F, Chezzi C. Respiratory tract infections and laboratory diagnostic methods: a review with a focus on syndromic panel-based assays. Microorg. 2022;10(9):1856.
21. 21. Minejima E, Wong-Beringer A. Implementation of rapid diagnostics with antimicrobial stewardship. Expert Revi Anti-Infect Ther. 2016;14(11):1065-75.
22. 22. Krause JC, Panning M, Hengel H, Henneke P. The role of multiplex PCR in respiratory tract infections in children. Deutsch Ärztebl Internat. 2014;111(38):639.
23. 23. Shiley KT, Lautenbach E, Lee I. The use of antimicrobial agents after diagnosis of viral respiratory tract infections in hospitalized adults: antibiotics or anxiolytics?. Infect Contr Hosp Epidemiol. 2010;31(11):1177-833.
24. 24. McCaig LF, Besser RE, Hughes JM. Antimicrobial-drug prescription in ambulatory care settings, United States, 1992–2000. Emerg Infect Dis. 2003;9(4):432.
25. 25. Spurling GK, Doust J, Del Mar CB, Eriksson L. Antibiotics for bronchiolitis in children. Cochr Datab System Revi. 2011(6).
26. 26. Vos LM, Bruning AH, Reitsma JB, Schuurman R, Riezebos-Brilman A, Hoepelman AI, et al. Rapid molecular tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review of diagnostic accuracy and clinical impact studies. Clin Infect Dis. 2019;69(7):1243-53.
27. 27. Jain N, Lodha R, Kabra S. Upper respiratory tract infections. Ind J Pediatr. 2001;68(12):1135-8.
28. 28. Vashishtha VM. Current status of tuberculosis and acute respiratory infections in India: much more needs to be done! Ind Pediatr. 2010;47(1):88-9.
29. 29. Weinstein AJ. Respiratory tract infections caused by Haemophilus influenzae in adults. Infection. 1987;15:S109-12.
30. 30. Pajapti B, Talsania N, Sonaliya KN. A study on prevalence of acute respiratory tract infections (ARI) in under-five children in urban and rural communities of Ahmedabad district, Gujarat. Natl J Community Med. 2011;2(02):255-9.
31. 31. Acharya D, Prasanna KS, Nair S, Rao RS. Acute respiratory infections in children: A community based longitudinal study in south India. Ind J Public Heal. 2003;47(1):7-13.
32. 32. Deb SK. Acute respiratory disease survey in Tripura in case of children below five years of age. J Indian Med Assoc. 1998;96(4):111-6.
33. 33. Kumar SG, Majumdar A, Kumar V, Naik BN, Selvaraj K, Balajee K. Prevalence of acute respiratory infection among under-five children in urban and rural areas of puducherry, India. J Natu Sci Biol Medi. 2015;6(1):3.
34. 34. Desai M, Buff AM, Khagayi S, Byass P, Amek N, van Eijk A, et al. Age-specific malaria mortality rates in the KEMRI/CDC health and demographic surveillance system in western Kenya, 2003–2010. PloS one. 2014;9(9):e106197.
35. 35. Jain S, Finelli L. Community-acquired pneumonia among US children. N Eng J Medi. 2015;372(22):2167-8.
36. 36. George M, Ahmad SQ, Wadowski S. Community-acquired pneumonia among U.S. Children. N Engl J Med. 2015;372:2166-7.
37. 37. Everard ML. Paediatric respiratory infections. Euro Respir Rev. 2016;25(139):36-40.
38. 38. Principi N, Zampiero A, Gambino M, Scala A, Senatore L, Lelii M, et al. Prospective evaluation of rhinovirus infection in healthy young children. J Clin Virol. 2015;66:83-9.
39. 39. Chonmaitree T, Alvarez-Fernandez P, Jennings K, Trujillo R, Marom T, Loeffelholz MJ, et al. Symptomatic and asymptomatic respiratory viral infections in the first year of life: association with acute otitis media development. Clin Infect Dis. 2015;60(1):1-9.
40. 40. Rhedin S, Lindstrand A, Hjelmgren A, Ryd-Rinder M, Öhrmalm L, Tolfvenstam T, et al. Respiratory viruses associated with community-acquired pneumonia in children: matched case-control study. Thorax. 2015;70(9):847-53.
41. 41. Shi T, McLean K, Campbell H, Nair H. Aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta–analysis. J Glob Heal. 2015;5(1):1.