1Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
2Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India.
3University of Alabama, Birmingham, USA.
Abstracts of the papers presented at the XVIII National Conference of Indian Virological Society at Post Graduate Institute of Medical Education and Research, Chandigarh, India, December 11–13, 2008.
Acute lower respiratory tract infections (ALRI) are major cause or mortality in children worldwide with similar clinical presentation. Available diagnostic methods have variable sensitivity and specificity, are labor intensive and time consuming. We aimed to optimize and evaluate in-house multiplex PCR for the detection of seven major ALRI causing viruses viz. Respiratory Syncytial Virus, Parainfluenza viruses (PIV1–3), influenza A and B viruses (Inf A and B) and newly discovered human Metapneumovirus (hMPV). Seven sets of primers were designed from Nucleocapsid gene (N) for RSV, hMPV, and PIV1–3 and Matrix gene (M) for Influenza viruses. Standard strains of all viruses were propagated in tissue culture and presence of virus was confirmed by indirect Immunofluorescence (IFA). A two tube multiplex PCR (mPCR) assay was standardized with RSV, Inf A and B viruses in one tube and PIVs and hMPV in another using the RNA extracted from tissue culture grown standard strains of viruses. Standardized mPCR was applied on 301 clinical samples viz. nasopharyngeal aspirates (NPAs) from children <5 years visiting AIIMS Pediatrics OPD with ALRI over a period of two years. RNA from NPAs was extracted from samples by RNeasy kit (Qiagen) and cDNA was synthesized using random hexamers. Specificity was tested by amplification of RNA from heterologus viruses and sensitivity by comparing with known TCID50 of standard strains of each virus. mPCR was found to be specific for each virus. Sensitivity of two tube mPCR assay was 0.1 TCID50 for RSV and Influenza A, 1TCID50 for hMPV, 10 TCID50 for PIV1, PIV2 and PIV3, and Influenza B. Of 301 NPAs, mPCR detected 86 single virus infections and 20 mixed infections (total 106; 35% of total samples collected). Of single virus infections, RSV was most commonly detected (20%) followed by PIVs (16%), hMPV (3.7%) and influenza A viruses (3%) of all the samples. The presence of co-infection with two or more viruses was observed in 6.6% of the total children included in the study. Of all the mixed virus infection combinations, PIV 2 with PIV3 was detected most commonly (11% of positives). mPCR is a rapid, specific, sensitive and less labor intensive method of testing for presence of respiratory viruses and is well suited for epidemiological studies in developing countries.
