title: Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases.
abstract: OBJECTIVE
  Influenza epidemics and periodic pandemics occur worldwide resulting in significant mortality, morbidity and economic loss. There is need for a sensitive, rapid and cost-effective assay to detect, type and sub-type influenza viruses, as cell culture has a long turnaround time.
  
  
  METHODS
  Nasopharyngeal swabs were collected from patients presenting with influenza-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 influenza A using primers for matrix gene and for influenza B using non-structural gene (NS) primers. All influenza 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 influenza by RT-PCR. Further typing analysis revealed 86 (63.2%) were typed as influenza A and 47 (34.5%) as influenza B viruses and 3 (2%) samples showed dual infection with influenza A and B. Of the 86 influenza A positive samples 48 (55.8%) were identified as seasonal influenza A/H1N1, 22 (25.6%) as A (H1N1) pdm09 and 16 (18.6%) as A/H3N2. Comparison of influenza positivity using virus culture revealed that only 97/136 (71.3%) were influenza positive. Sensitivity of viral detection was lowest for seasonal A/H1 (26/48; 54%), followed by H3N2 (11/16; 68.7%) and influenza B (38/47; 80.8%); all influenza A/H1N1pdm09 viruses were detected by both methods.
  
  
  CONCLUSIONS
  RT-PCR is a sensitive, low cost and rapid screening test for diagnosing influenza infection during epidemics and pandemics. mRT-PCR increased the detection rates for influenza by 28.6% as compared with virus isolation and thus is a useful assay in both diagnostic and epidemiological settings in resource poor countries.
authors.forename: S
authors.initials: S
authors.lastname: Dhakad
authors.forename: P C
authors.initials: PC
authors.lastname: Mali
authors.forename: S
authors.initials: S
authors.lastname: Kaushik
authors.forename: A A
authors.initials: AA
authors.lastname: Lal
authors.affiliation: INCLEN Trust International , New Delhi, India.
authors.forename: S
authors.initials: S
authors.lastname: Broor
mesh_terms: D003365:Costs and Cost Analysis; D017931:DNA Primers / Q000235:genetics; D003955:Diagnostic Tests, Routine / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009305:Nasopharynx / Q000821:virology; D009439:Neuraminidase / Q000235:genetics; D009975:Orthomyxoviridae / Q000145:classification* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D014776:Virus Cultivation / Q000379:methods
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C108309:NS protein, influenza virus; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase