Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases.
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Date
2015-01
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Abstract
Purpose: Infl uenza epidemics and periodic pandemics occur worldwide resulting in signifi cant mortality, morbidity
and economic loss. There is need for a sensitive, rapid and cost-effective assay to detect, type and sub-type
infl uenza viruses, as cell culture has a long turnaround time. Materials and Methods: Nasopharyngeal swabs
were collected from patients presenting with infl uenza-like illness (ILI) at AIIMS OPD and Primary Health
Centre Ballabhgarh (Haryana). From June 2007 to January 2009 and then from September to November 2009, of
1567 specimens collected, 544 were randomly selected and were tested by virus culture using Madin-Darby Canine
Kidney (MDCK) cells and by reverse transcription polymerase chain reaction (RT-PCR) for infl uenza A using
primers for matrix gene and for infl uenza B using non-structural gene (NS) primers. All infl uenza A positives were
sub-typed using primers for HA and NA genes of A/H1, A/H3. A separate multiplex RT-PCR having primers from
matrix and HA genes of pandemic A (H1N1) pdm09 viruses was carried out on samples collected after September
2009. Results: Of the 544 samples, 136 (25%) were positive for infl uenza by RT-PCR. Further typing analysis revealed
86 (63.2%) were typed as infl uenza A and 47 (34.5%) as infl uenza B viruses and 3 (2%) samples showed dual infection
with infl uenza A and B. Of the 86 infl uenza A positive samples 48 (55.8%) were identifi ed as seasonal infl uenza
A/H1N1, 22 (25.6%) as A (H1N1) pdm09 and 16 (18.6%) as A/H3N2. Comparison of infl uenza positivity using virus
culture revealed that only 97/136 (71.3%) were infl uenza positive. Sensitivity of viral detection was lowest for seasonal
A/H1 (26/48; 54%), followed by H3N2 (11/16; 68.7%) and infl uenza B (38/47; 80.8%); all infl uenza A/H1N1pdm09
viruses were detected by both methods. Conclusion: RT-PCR is a sensitive, low cost and rapid screening test for
diagnosing infl uenza infection during epidemics and pandemics. mRT-PCR increased the detection rates for infl uenza
by 28.6% as compared with virus isolation and thus is a useful assay in both diagnostic and epidemiological settings in
resource poor countries.
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Keywords
Infl uenza, Pandemic H1N1, Cell culture, Reverse transcription-polymerase chain reaction
Citation
Dhakad S, Mali P C, Kaushik S, Lal A A , Broor S. Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases. Indian Journal of Medical Microbiology. 2015 Jan-Mar ; 33 (1): 73-77.