High sensitivity detection of JC-virus DNA in postmortem brain tissue by in situ PCR.

Opportunistic infection of the central nervous system by human polyomavirus JC can cause a devastating disease, progressive multifocal leukoencephalopathy (PML). To gain new neuropathological insights into JC-virus (JCV) infection patterns in PML at the light microscopic level, the highly sensitive indirect in situ polymerase chain reaction (in situ PCR) was employed in up to 15-year old formalin-fixed and paraffin-embedded postmortem brain tissue derived from nine AIDS patients with PML. In situ PCR, in which target DNA is amplified intracellularly and detected by a specific labelled probe in morphologically intact tissue, was compared with conventional in situ hybridization (ISH). Validity was ensured by the inclusion of 13 controls. JCV detection with in situ PCR proved to be highly sensitive since in all nine brain samples the number of positive cells exceeded the ISH results by 2-3-fold. Whereas by routine staining the brain tissue of each individual patient showed regions with severe, mild or no involvement by PML, improved detection of JCV DNA by in situ PCR allowed a regrading into five different degrees of JCV infection. Significant myelin staining was observed, suggesting that cell-to-cell contact may not be the only means of virus spread but that new cells could also be infected by virus released after cell lysis. Furthermore, using in situ PCR hitherto unreported intracellular distribution patterns of JCV DNA in oligodendro- and astrocytes were observed by light microscopy.