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Influenza A and B are RNA-containing viruses that frequently infect humans. Currently, sensitive detection of these viruses requires fresh respiratory secretions and special facilities for culture. To facilitate diagnosis of influenza, the polymerase chain reaction (PCR) was used in the present studies to detect DNA produced by reverse transcription of influenzal RNA in vaccines, tissue culture fluids, and stored respiratory secretions. Primers were directed at targets on the highly conserved segment 7 (matrix gene) of influenza A (212-bp product) and B (365-bp product). The primers were completely type specific. Critical variables in the assay were the concentration of pleotropic salts used during preparation of samples, the use of carrier RNA and RNase inhibitors during sample preparation, and the use of optimum K+ and Mg2+ levels at each step. Studies of 33 patients with symptoms of viral respiratory infection whose nasal washes had been cultured and frozen for up to 1 year before assay showed that PCR provided type-specific detection of influenza with a sensitivity comparable to that of culture of the fresh secretions. The assay offers a powerful test for detection of devitalized influenza viruses and may be useful in both diagnostic work and epidemiological studies of influenza.

A solid-phase enzyme-linked immunosorbent assay (ELISA) with monoclonal secondary antibodies was used to detect matrix protein and nucleoprotein of influenza A. The sensitivity of the ELISA for highly purified A/Brazil nucleoprotein and matrix protein was 0.05 and 1.0 ng, respectively. Nasal washes from 10 of 20 adult subjects with culture-proven, naturally acquired infection caused by A/Brazil/11/78-like influenza virus were positive in the test, and 2 of 13 subjects with rhinovirus infection were falsely positive. To determine if ELISA results could be improved, nasal washes were obtained from 21 adult volunteers who had been inoculated intranasally with wild-type A/Korea/1/82 (five subjects) or A/Korea recombinants with matrix protein or RNA-2 protein of A/Ann Arbor/6/60 (16 subjects), and the nasal washes were processed by a variety of methods. Prompt addition of sodium azide to the nasal washes to limit bacterial growth, avoidance of freezing, and the use of an antiproteolytic agent all failed to improve ELISA results noticeably. Under the best conditions, ELISA was positive in only 12 of the 21 experimentally infected subjects and in 1 of 15 uninfected controls. Positive ELISA results in experimentally infected subjects correlated significantly with the titer of live virus in the nasal washes (r = +0.506; P less than 0.001). Detection of gradient-purified whole influenza virus or isolated core antigen in ELISA was inhibited by prior incubation with nasal washes, and the inhibitory activity was only partly decreased by heat treatment of the secretions. At present, the use of ELISA for detection of influenza antigens in respiratory secretions is not sufficiently sensitive or specific for routine laboratory diagnosis of influenza.

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