Improved detection of human influenza A and B viruses in respiratory tract specimens by hemi-nested PCR

Abstract

RT-PCR is the most sensitive assay for diagnosis of influenza, due to enhanced rapidity and sensitivity as compared to classical methods. Hemi-nested RT-PCR was developed, targeting NP gene for influenza A and NS gene for influenza B, based on a previous single round RT-PCR method. The new method was compared with the previous technique for analytical sensitivity and specificity, and was applied to clinical samples from the lower and upper respiratory tract. The analytical sensitivity of hemi-nested RT-PCR was 10 (influenza A) and 4 times (influenza B) higher than the previous method. A high specificity of the new hemi-nested RT-PCR assay was observed by using whole respiratory viruses. When applied to lower respiratory tract specimens, the new method showed an increased rate of positivity as compared to the previous technique (9.3% versus 0.7% for influenza A, and 0.9% versus 0.2% for influenza B). Screening of upper respiratory tract samples collected during the seasonal 2005-2006 outbreak indicated 26.4% and 5.8% positivity for influenza A and B, respectively. The results were confirmed by sequence analysis: apart from influenza B, both influenza A subtypes H3N2 and H1N1, associated with the seasonal outbreak, were detected.

Fig. 1

Detection of influenza A and B amplicons by hemi-nested and reference RT-PCR at limiting dilution of positive control plasmids containing the target sequences as the insert (influenza A plasmid concentration = 5 × 10¹⁰ copie/ml; influenza B plasmid concentration = 2.5 × 10¹¹ copie/ml).

Fig. 2

Monthly distribution of lower respiratory tract samples positive to influenza A and B by hemi-nested RT-PCR, during the period March 2004–May 2005. Continuous line: total number of samples collected. Grey bars: influenza A-positive sample. Black bars: influenza B-positive samples.