USE OF PCR-ENZYME IMMUNOASSAY FOR IDENTIFICATION OF INFLUENZA A VIRUSMATRIX RNA IN CLINICAL-SAMPLES NEGATIVE FOR CULTIVABLE VIRUS

Influenza A virus infections are a major cause of morbidity and mortal ity worldwide. Standard diagnostic methods either are not efficient in identifying infected individuals in a timely manner or lack sensitivi ty. We developed a PCR-enzyme immunoassay (PCR-EIA) for the detection of influenza A virus RNA in respiratory secretions. A reverse transcri ption PCR was performed with oligonucleotide primers directed at a hig hly conserved area of the influenza A matrix gene. Amplified DNA was i dentified by hybridization in solution to a nested biotinylated RNA pr obe and quantitated in an EIA. PCR-EIA detected small quantities of RN A from the three prevalent subtypes of human influenza A virus. Influe nza B and C, parainfluenza, measles, mumps, and respiratory syncytial viruses tested negative. The potential efficiency of PCR-EIA for use i n clinical diagnosis was determined by testing 90 nasal wash specimens obtained daily over a 10-day period from nine human volunteers infect ed with influenza A virus. Thirty-seven of the postinfection samples h ad detectable influenza A virus RNA by PCR-EIA, whereas only 26 postin fection samples were positive by culture. PCR-EIA was particularly eff icient for the identification of influenza A virus in samples obtained more than 4 days after infection. Seventeen of 45 such samples were p ositive, whereas virus was cultivated from 4 samples (P < 0.00005). Al l preinfection samples from volunteers subsequently infected with infl uenza A virus were negative by PCR-EIA, as were samples from a volunte er infected with parainfluenza virus type 3. Nucleic acid amplificatio n techniques represent important tools for the timely and sensitive di agnosis of influenza A virus infections and, therefore, their manageme nt and control.