McSharry James J, McDonough Ann C, Olson Betty A, Drusano George L
Center for Immunology and Microbial Disease and Clinical Research Initiative, Albany Medical College, Albany, New York 12208, USA.
Clin Diagn Lab Immunol. 2004 Jan;11(1):21-8. doi: 10.1128/cdli.11.1.21-28.2004.
A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.
已开发出一种流式细胞术（荧光激活细胞分选仪 [FACS]）检测方法，用于分析甲型和乙型流感病毒野生型及耐药实验室毒株以及临床分离株对神经氨酸酶（NA）抑制剂奥司他韦羧酸盐、扎那米韦和帕拉米韦的药物敏感性。通过FACS检测方法可以轻松确定野生型流感病毒以及血凝素（HA）和/或NA基因发生突变而对一种或多种NA抑制剂耐药的流感病毒的药物敏感性。通过FACS检测方法确定的使病毒感染细胞数量或空斑形成单位（PFU）数量减少50%的药物浓度，与通过耗时且 labor-intensive 的病毒产量减少检测方法获得的浓度相似。NA抑制（NAI）检测证实了FACS和病毒产量检测所显示的NA基因发生突变的耐药流感病毒的耐药模式。然而，只有FACS和病毒产量检测能检测到HA基因发生突变但NA基因未发生突变的对NA抑制剂耐药的流感病毒。FACS检测比病毒产量检测更快速且 labor-intensive 程度更低，并且同样具有定量性。FACS检测可确定HA或NA基因发生突变的流感病毒的药物敏感性，这使得该检测方法在测量流感病毒对NA抑制剂的体外敏感性方面比NAI检测更具广泛用途。然而，由于只有NA基因发生突变且导致对NA抑制剂耐药的病毒才与临床耐药相关，因此这种体外检测方法不应在临床环境中用于确定对NA抑制剂的耐药性。该检测方法可能有助于确定其他对甲型和乙型流感病毒有效的化合物的体内敏感性。
