Negative-staining Electron Microscopy of the Urine for the Detection of Polyomavirus Infections

Abstract

Negative-staining electron microscopy (EM) has played a pivotal role in diagnostic virology. It is a rapid technique for viral detection in the urine and can provide an easy means for monitoring viral activity and productive infections. EM of urine for the detection of polyomaviruses has hitherto not been systematically evaluated as a screening tool for renal transplant patients at risk for BK polyomavirus nephropathy (BKN). Here, the authors discuss technical aspects of negative-staining EM of urine (n = 76 samples) and present a simple and rapid protocol for the semiquantitative evaluation of patient samples. In two patient populations (either with (n = 15 samples) or without (n = 15 samples) an established diagnosis of BKN), EM results were compared with two previously established techniques for monitoring polyomavirus activation: (1) cytology for the quantitation of decoy cells, and (2) quantitative PCR assays for the detection of BK virus DNA load levels. In both patient groups, the dynamics of decoy cell shedding by urine cytology closely paralleled free viral particle shedding by EM, and viral load levels as measured by PCR. A trend toward higher readings was observed in patients with BKN (median values, control versus BKN groups: decoy cells 21 versus 50/slide; free virions by EM: 32 versus 66 viral particles/10 high-power fields; PCR: 3.5 × 108 versus 5.4 × 108 BK virus copies/ml; all differences not statistically significant). The authors conclude that negative-staining EM and the semiquantitative assessment of free viral particles in the urine can be a useful clinical method to identify patients at increased risk for BKN. EM can be used alone or in combination with urine cytology or PCR assays.