Real time RT-PCR for the detection and sub typing of Seasonal Influenza viruses in south India.
Session III – Moorthy, Mahesh – Young Investigator Applicant
Title of Contribution: Real time RT-PCR for the detection and sub typing of Seasonal Influenza viruses in south India.
Author(s): Mahesh Moorthy, Maria Thomas, Valsan Varghese*, Dilip Mathai#, Asha Mary Abraham
Affiliation(s): Department of Clinical Virology, Child Health* and Medicine#, Christian Medical College, Vellore, India
Abstract: Seasonal influenza viruses are an important cause of morbidity and mortality around the world. Surveillance for influenza viruses in India has been undertaken only for the last few years.The method of choice for surveillance has been isolation of influenza viruses using MDCK cells and identification of viral isolates with reference anti-sera provided by the WHO.The main drawbacks of this cell culture-based system are that it is labor intensive and results are delayed by up to 3 weeks.
We used a real time reverse transcriptase polymerase chain reaction (RT-PCR) based on Taqman chemistry for the detection of influenza viruses.
Methods: Samples were collected from children and adults as part of an ongoing influenza surveillance study. Subjects with either sudden onset of fever > 101°F, body ache, and other symptoms of respiratory infection including - cough, sore throat, breathlessness, expectoration, presence of crepitations, wheeze or other lower respiratory signs were recruited. Nasal or throat swabs were collected and viral RNA extracted. The real time assay designed by the CDC, Atlanta, consisting of 4 uniplex real time RT-PCR reactions for detection of influenza A or B viruses (typing) and detection of influenza A subtype H1 or H3 (sub-typing) was used. An RT-PCR for the detection of human RNase P served as an internal control. Probes labelled with 6-FAM were used and assays performed on a Rotor Gene real time PCR system. RNA extracts were initially tested for influenza A, influenza B and RNP. Positive samples were then subjected to repeat extraction and real time RTPCR for the detection of Influenza A, B, H1 and H3.
Results: Fifteen of a total of 192 samples tested with the preliminary RT-PCR assay were positive. RNP RT-PCR was negative for 1 sample which was excluded from further analysis. Of the 15/191 positive samples, 6 were weak positive with Ct values of around 40. A repeat extraction was performed on all 15 along with the second assay for Influenza sub-typing. Of the 15 samples that were initially positive, 9 were found to be repeat positive. All 9 samples were Influenza A subtype H3 with Ct values raging from 24 to 36. On examination of the clinical features and management of patients recruited in this study, it was found that 7/9 patients positive for influenza and 152/182 patients negative for influenza received either parenteral or oral antibiotic therapy that was initiated on admission. Since the turnaround time of real time RT-PCR is 1-2 days, use of this testing methodology on a routine basis will ensure administration of antibiotics only where indicated. Patients with influenza presented predominantly with fever (100%), cough (77.8%), nasal discharge (66.7%) and lower chest indrawing (44.4%). It was noteworthy that influenza positive patients in general presented with a range of 6 to 10 out of 23 signs and symptoms assessed during sample collection, but influenza negative patients presented with a wider range of between 2 and 15 signs and symptoms.
Summary: The real time RT-PCR for influenza can be used for the rapid testing of patients presenting with respiratory illness. This would be useful in avoiding inappropriate antibiotic treatment and determining the epidemiology of influenza in the population. This approach can further be used to study the prevalence of other respiratory viral pathogens in the community.
