Human influenza A virus (H5N1) detection by a novel multiplex PCR typing method.

We report the use of ResPlex III for genotyping influenza A viruses. The performance characteristics of the assay with regard to H5N1 are further evaluated. The ResPlex system incorporates a novel multiplex PCR technology, target-enriched multiplex PCR, to simultaneously amplify multiple molecular targets in one reaction. The ResPlex III assay targets the H1, H2, H3, H5, H7, H9, N1, and N2 genes from the influenza A virus as well as the NS genes from influenza A (NSA) and B (NSB) viruses, providing detection and genotyping of influenza A and B viruses. The analytical sensitivities for detecting the H5, N1, and NSA genes were 1, 10(-1), and 10 50% tissue culture infectious doses/200 microl/reaction, respectively. A total of 217 sequential clinical samples including 14 samples with human H5N1 infections were tested by the ResPlex III assay, and the results were compared to a reference standard combined with results of viral culture and conventional reverse transcriptase and real-time PCR. The clinical sensitivity and specificity for detecting H5N1 were 93.3% and 100%, respectively, indicating that different subtypes of influenza A virus can be quickly and correctly identified using the ResPlex III genotyping approach.

Evaluation of a Digene-recommended algorithm for human papillomavirus low-positive results present in a "retest zone".

The Digene Hybrid Capture 2 (hc2) high-risk human papillomavirus (HPV) DNA test (Digene, Gaithersburg, MD) is widely used for triage of women with atypical squamous cells of undetermined significance. Results in a "retest zone" (weakly positive tests) are repeated up to 2 times according to the Digene-recommended algorithm. We studied 56 cervical samples in the retest zone. Specimens were tested by a multiplex polymerase chain reaction (PCR)-based genotyping assay, and relevant cytopathologic results were reviewed. Digene results were compared with a reference standard that combined PCR genotyping and cytopathology results. The first repeated Digene assay yielded a sensitivity of 85.2% and a specificity of 62.1% with false-positive and false-negative rates of 40.0% and 15.4%, respectively. The 22 negative samples underwent a second retest and 18 (82%) were negative by the reference standard. The combined first and second retest sensitivity, specificity, and predictive values remained unchanged from the first retest alone. Repeating specimens in the retest zone is necessary, but a second retest does not offer advantages over the first retest.

Simultaneous amplification and identification of 25 human papillomavirus types with Templex technology.

The majority of existing human papillomavirus (HPV) genotyping assays are based on multiplex PCR using consensus or degenerate primers. We developed a Templex HPV assay that simultaneously detects and identifies 25 common HPV genotypes in a single-tube reaction using type-specific primers for the HPV-specific E6 and E7 genes. The analytical sensitivities of the Templex assay for HPV type 16 (HPV-16), -18, and -56 were 20, 100, and 20 copies per reaction mixture, respectively. The Templex assay provides semiquantitative information on each type when multiple HPV types coexist in one reaction. We tested 109 clinical cervical specimens previously evaluated with the Digene HC2 high-risk HPV DNA test and found 95.4% concordance between the assay results. The Templex assay provided type-specific results and found multiple types in 29.2% (14 of 48) of high-risk HPV-positive samples. The entire Templex procedure, including DNA extraction, can be completed within 5 hours, providing a rapid and reliable diagnostic tool for HPV detection and typing that is amenable to automation.