Dual qualitative-quantitative nested PCR for detection of JC virus in cerebrospinal fluid: High potential for evaluation and monitoring of progressive multifocal leukoencephalopathy in AIDS patients receiving highly active antiretroviral therapy

JC polyomavirus (JCV) is the causative agent of progressive multifocal leuk oencephalopathy (PML), a central nervous sa stem infection that mainly affe cts AIDS patients, The extensive application of highly active antiretrovira l therapy (HAART) is leading to the appearance of "long-term" survival PML patients. A reliable and feasible qualitative-quantitative test for both th e detection of JCV and follow-up of its viral burden in this emerging group of patients is clearly required. With this aim, a dual qualitative-quantit ative nested PCR is presented in this study for the analysis of JCV DNA in cerebrospinal fluid (CSF), Two newly designed internal controls, one compet itive and the other noncompetitive, have been constructed to adapt this PCR to either measure the JCV burden or to allow a highly confident determinat ion of JCV presence or clearance. The analytical sensitivity of the techniq ue allows the detection of 0.01 fg (three genomes) of JCV DNA. Its qualitat ive application has been evaluated by analyzing single CSF samples from a g roup of 17 patients with PML and a control group of 20 patients with divers e neurological conditions other than PML, yielding sensitivity and specific ity values of 100 and 90%, respectively. The quantitative application has b een evaluated in vitro in blind tests with samples including serial dilutio ns of JCV, and in all cases the samples were successfully ordered consideri ng the JCV titer, The dual quantitative, e qualitative application offered by this nested PCR may provide an answer to the new requirements for evalua ting and finely monitoring PML in AIDS patients receiving HAART.