Improved detection of JC virus in AIDS patients with progressive multifocal leukoencephalopathy by T-antigen specific fluorescence resonance energy transfer hybridization probe real-time PCR: evidence of diverse JC virus genotypes associated with progressive multifocal leukoencephalopathy in Southern Africa

Abstract

JC virus (JCV) causes progressive multifocal leukoencephalopathy under conditions of immunosuppression, especially associated with HIV. Despite the high prevalence of HIV-1 infection, few cases of progressive multifocal leukoencephalopathy have been reported and only a small number of JCV strains have been characterized in AIDS patients in Southern Africa. Diagnosis of progressive multifocal leukoencephalopathy through PCR detection of JCV DNA in CSF may result in false negative results if variable regions of the genome are targeted. Characterization of circulating JCV genotypes in Southern Africa may assist with designing appropriate diagnostic assays and characterizing the molecular epidemiology of JCV in African AIDS patients. In this study, a new real-time PCR using fluorescence resonance energy transfer hybridization probes targeting the conserved large T-antigen was developed and compared to a conventional nested PCR targeting the variable VP1 region. Validation against known JCV positive specimens confirmed its specificity while amplification of a serial dilution of JCV control DNA suggests that the new real-time PCR can detect lower concentrations of JCV than the VP1 nested PCR. Investigation of CSF specimens from 44 suspected progressive multifocal leukoencephalopathy patients suggest that the new assay is more sensitive being able to detect JCV in 12 specimens versus 5 specimens with the VP1 nested PCR. Sequence analysis confirmed that the T-antigen region is completely conserved while phylogenetic analysis of the five VP1 products identified two genotype 3, one genotype 1 and two genotype 4 strains, the latter two identified for the first time in South African AIDS patients.