Profile of respiratory pathogens causing acute respiratory infections in hospitalised children at Rajasthan a 4 year's study.

Introduction: Various pathogens cause respiratory tract infections in children of <5 years of age causing severe morbidity and mortality. The profile of causative agents varies from place to place. Aims: The objectives of our study were to detect the profile and trends of respiratory pathogens causing acute respiratory tract infection in children using a custom multiplex real-time polymerase chain reaction (RT-PCR) and to develop a diagnostic algorithm. Materials and Methods: A total of 997 children with clinical manifestations of respiratory infections were included in the study. Their nasopharyngeal aspirate and throat swab samples were subjected to nucleic acid extraction followed by multiplex RT-PCR for eighteen viruses and six bacteria. Statistical Analysis Used: Chi-square test was employed to study the P value of different viruses and bacteria. Results: A total of 765 (76.73%) samples were found to be positive for one of the respiratory pathogens. Viruses were detected in 598 (59.98%) and bacteria in 167 (41.85%) samples, respectively. The prevalence of single and co-infections among viruses and bacteria were 77.76% and 22.24%, 81.44% and 18.56% each, respectively. Respiratory syncytial virus (RSV) A/B and Streptococcus pneumoniae were the most predominant pathogens detected in the study and were associated with lower respiratory tract infections. Conclusion: RSV and S. pneumoniae were the most common pathogens detected, higher prevalence was observed in children <1 year of age. Viruses were predominant during winter months. The study helped to prepare diagnostic algorithm which will help in reducing diagnostic costs. However, further studies are required to assess whether viruses are bystander or real pathogens and include larger panel of bacteria and viruses for diagnosis.

Trends of respiratory syncytial virus sub-types in children hospitalised at a tertiary care centre in Jaipur during 2012-2014.

Respiratory syncytial virus (RSV) causes high mortality and morbidity in infants. The study was planned to determine the trends of RSV sub-types in hospitalised children. Nasopharyngeal aspirate and throat swabs were collected from the hospitalised children up to 5 years of age. Viral nucleic acid was extracted using easyMAG automated extraction system, and real-time reverse transcription polymerase chain reaction was performed. Total positivity for RSV was found to be 25.40%, predominantly for RSV B (20.03%), followed by RSV A (2.90%) and RSV AB mixed infections (2.47%). Palivizumab prophylaxis can be planned to be given to infants from post-monsoon to end of winter.

Evaluation of custom multiplex real - time RT - PCR in comparison to fast - track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses.

BACKGROUND: Severe acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD). METHODS: Nasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples. RESULTS: Custom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 and 94.66 - 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more. CONCLUSION: The custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.

Distribution and Trends of Human Parainfluenza Viruses in Hospitalised Children.

OBJECTIVE: To study the distribution of Human Parainfluenza viruses (HPIV) 1-4 and their trends in children ≤5 y of age, hospitalised at a tertiary care centre, Jaipur and co-infection with other respiratory viruses. METHODS: Nasopharyngeal aspirate and throat swabs were collected and processed for extraction of nucleic acid using automated extraction system and real time RT-PCR was performed using primers and probes specific to HPIV 1-4 and other respiratory viruses on 743 samples. RESULTS: Total positivity for Parainfluenza viruses 1-4 was found to be 69/743 (9.28 %), of which 50/533 (9.38 %) were boys and 19/210 (9.05 %) girls. Predominance of HPIV- 3 was observed [41/743 (5.52%)] followed by HPIV-1 in 13/743 (1.75%), HPIV-4 in 10/743 (1.34%) and HPIV-2 in 5/743 (0.67%) patients. Maximum positivity was observed in age group 25-36 mo (12.98%) followed by 13-24 mo group (11.96%). HPIVs were found to be circulating round the year and each year. Co-infections with other respiratory viruses were observed in 22/69 (31.88%) of HPIV positive patients. CONCLUSIONS: All the four types of HPIV were found to be circulating in the index population during all the three years, predominantly during post monsoon and winter seasons. HPIV vaccination should be targeted for all types.

Viruses causing severe acute respiratory infections (SARI) in children ≤5 years of age at a tertiary care hospital in Rajasthan, India.

BACKGROUND & OBJECTIVES: Severe acute respiratory infection (SARI) is one of the leading causes of death among children worldwide. As different respiratory viruses exhibit similar symptoms, simultaneous detection of these viruses in a single reaction mixture can save time and cost. The present study was done in a tertiary care children's hospital for rapid identification of viruses causing SARI among children less than or equal to five years of age using multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) kit. METHODS: A total of 155 throat swabs were collected from equal number of children suspected to have SARI and processed for extraction of nucleic acids using automated extraction system. Multiplex real-time RT-PCR was done to identify the viruses in the samples. RESULTS: The overall positivity for viruses in the study was found to be 72.9 per cent with a co-infection rate of 19.5 per cent. Human metapneumovirus (HMPV) was the predominant virus detected in 25.7 per cent children followed by influenza A (H1N1)pdm09, human rhinovirus (HRV) and human adenovirus (HAdV) in 19.9, 11.0 and 8.8 per cent children, respectively. The HMPV was at its peak in February 2013, HAdV showed two peaks in March-April, 2012 and November 2012-March 2013 while HRV was detected throughout the year. INTERPRETATION & CONCLUSIONS: Multiplex real-time PCR helped in rapid identification of viruses. Seventeen viruses were detected in SARI cases with overall positivity of 72.9 per cent. HMPV was the most predominant virus. However, for better clinico-virological correlation, studies are required with complete work up of all the aetiological agents, clinical profile of patients and treatment outcome.