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High sensitivity detection of JC-virus DNA in postmortem brain tissue by in situ PCR
I W Samorei1, M Schmid, M Pawlita
1Department of Psychiatry, Ruprecht-Karls-Universität, Heidelberg, Germany.
Abstract:
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.
Insights
Human polyomavirus JC (JCV) causes progressive multifocal leukoencephalopathy (PML). In situ PCR revealed higher JCV detection in AIDS patient brains, offering new insights into infection patterns and spread.
Area of Science:
- Neurovirology
- Neuropathology
- Molecular Biology
Background:
- Progressive multifocal leukoencephalopathy (PML) is a severe central nervous system disease caused by opportunistic human polyomavirus JC (JCV) infection.
- Understanding JCV infection patterns is crucial for diagnosing and managing PML, particularly in immunocompromised individuals like AIDS patients.
- Current diagnostic methods may not fully capture the extent of JCV infection in affected brain tissue.
Purpose of the Study:
- To investigate JC-virus (JCV) infection patterns in the brain tissue of AIDS patients with PML using enhanced neuropathological techniques.
- To compare the sensitivity and diagnostic utility of indirect in situ polymerase chain reaction (in situ PCR) with conventional in situ hybridization (ISH) for JCV detection.
- To gain new insights into the cellular distribution and spread of JCV within the central nervous system at the light microscopic level.
Main Methods:
- Indirect in situ PCR was performed on formalin-fixed, paraffin-embedded postmortem brain tissue from nine AIDS patients diagnosed with PML.
- The sensitivity of in situ PCR for JCV DNA detection was compared with conventional in situ hybridization (ISH).
- Thirteen control samples were included to ensure the validity of the detection methods.
Main Results:
- In situ PCR demonstrated significantly higher sensitivity, detecting 2-3 times more JCV-positive cells compared to ISH across all nine patient samples.
- Improved detection by in situ PCR enabled a more precise grading of JCV infection severity, categorizing involvement into five degrees.
- Unreported intracellular distribution patterns of JCV DNA were observed in oligodendrocytes and astrocytes, suggesting infection spread through cell lysis in addition to cell-to-cell contact.
Conclusions:
- Indirect in situ PCR is a highly sensitive method for detecting JCV DNA in PML brain tissue, surpassing conventional ISH.
- The findings provide novel neuropathological insights into JCV infection dynamics, including potential spread mechanisms and cellular tropism.
- Enhanced detection capabilities offered by in situ PCR can improve the assessment of PML severity and inform future research on JCV pathogenesis.