Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase.

BACKGROUND: Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed. OBJECTIVES: We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene. STUDY DESIGN: Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza. RESULTS: 121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected. CONCLUSIONS: We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms.

Diagnosing herpesvirus infections by real-time amplification and rapid culture.

Procedures using real-time technique were developed to demonstrate the presence of herpes simplex virus type 1 (HSV-1) and HSV-2, varicella zoster virus (VZV), and cytomegalovirus (CMV) in miscellaneous clinical specimens. The assays were compared to rapid culture using centrifugation followed by detection with monoclonal antibodies. A total of 711 consecutive samples were collected from different patient groups. Throat swabs were obtained from transplant patients; dermal or oral specimens were collected from patients suspected for VZV or HSV infection. Genital specimens were taken from patients who attended the Clinic for Sexually Transmitted Diseases at the Dijkzigt Hospital Rotterdam presenting with symptoms of a primary genital ulcer. Nucleic acid extraction was carried out using a MagnaPure LC instrument. The amplification steps were performed on the ABI Prism 7700 sequence detection system. To monitor the process of extraction and amplification, a universal control consisting of seal herpesvirus type 1 (PhHV-1) was added to the clinical specimens. By culture 127 of 668 (19%) samples were positive for HSV-1, 72 of 668 (10.8%) specimens were positive for HSV-2, and 17 of 366 (4.6%) were positive for VZV. Using real-time amplification the numbers of positive specimens were 143 of 668 (21.4%), 97 of 668 (14.5%), and 27 of 366 (7.4%), respectively. Eighty-six specimens were tested for CMV, 12 (14.0%) were positive by culture, and 17 (19.8%) were positive by real-time PCR. The clinical data of the patients with discrepant results were reviewed thoroughly. In all cases the patients with only real-time PCR-positive results could be considered as truly infected. We concluded that the real-time amplification technique is suitable for the detection of human herpesvirus infection. It offers a semiquantitative and reliable assay with a quick result that is more sensitive than rapid culture, especially for the diagnosis of HSV-2 and VZV infections.