Nucleic acid dipstick test for molecular diagnosis of pandemic H1N1

Abstract

A new nucleic acid amplification-based rapid test for diagnosis of pandemic influenza (H1N1) 2009 virus was developed. The molecular test for pandemic H1N1, SAMBA (simple amplification-based assay), is based on isothermal amplification and visual detection on a dipstick characterized by high sensitivity, high specificity, a short turnaround time, and minimal technical requirements. The amplification step is monitored with an internal control to ensure correct interpretation of test results. The clinical performance of this assay was evaluated using blinded RNA samples extracted from nasal/throat swab specimens from 262 patients exhibiting influenza-like illness. Compared with the United Kingdom National Standard Method, based on quantitative reverse transcription-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the new assay were 95.3% (95% confidence interval, 88.5 to 98.7%), 99.4% (95% confidence interval, 96.9 to 99.9%), 98.8% (95% confidence interval, 93.5 to 99.9%), and 97.8% (95% confidence interval, 94.4 to 99.4%), respectively. The SAMBA for pandemic H1N1 provides a new technology that could potentially facilitate timely diagnosis and management of infected individuals, thereby informing decision making with regard to patient isolation during a pandemic outbreak.

FIG. 1.

Detection of pandemic (H1N1) 2009 virus on the dipstick. After amplification of the influenza viral genome, the amplification product was visually detected on the dipstick by naked eye and the signal strength can be scored according to the scoring chart. (a) Reference scoring chart showing signal strength from 0.5 to 5 in 0.5-point increment. (b) Samples of test results on the dipsticks showing strong positive, intermediate positive, and negative (from left to right) samples. The upper line is the internal control line, which should be present in all tests to validate the test run; the lower line is the test line indicating the presence of the pandemic (H1N1) 2009 viral genome in the samples.

FIG. 2.

Correlation of the dipstick signal for the SAMBA for pandemic H1N1 and the C_T value for qRT-PCR with clinical samples. Samples that tested positive by both the SAMBA for pandemic H1N1 and qRT-PCR (○), those that tested initially negative but subsequently positive by the SAMBA (•), and those that tested negative by the SAMBA but positive by qRT-PCR (▵) are indicated. The C_T value is inversely proportional to the viral load. A significant inverse correlation was apparent between the two sets of results (r = −0.68, P < 0.0001).