Absence of JC polyomavirus in stool samples of patients with multiple sclerosis despite high anti-JCV antibodies in serum

Background Natalizumab is an effective treatment for relapsing multiple sclerosis (MS). During therapy, individuals are at increased risk of developing progressive multifocal leukoencephalopathy (PML). So far, the relevant reservoir for PML-type JC polyomavirus (JCV) remains elusive. We here tested if the detection of JCV-DNA in stool of persons with MS treated with natalizumab could be a future tool for PML risk assessment. Methods The presence of JCV-DNA in stool, urine, and whole blood of MS patients treated with natalizumab and known serum anti-JCV antibodies index values (IV) was studied. Different DNA extraction methods, real-time (RT) and droplet digital (dd) PCR techniques were compared. JCV isolates were screened for PML-associated variants by sequencing. Results Thirty MS patients treated with natalizumab were screened. For 21 patients, blood, stool, and urine samples were available. These patients were stratified according to their serum anti-JCV antibody IV (high (>1.5, n=12); medium (1.5-0.9, n=2); low (<0.9, n=1); negative (n=6)). JCV-DNA could not be detected in the whole blood or stool samples. Four urine samples had measurable JCV-DNA, ranging from 1.71*104-1.07*108 international units (IU)/mL detected by RT-PCR, corresponding to 4.62*104-9.85*106 copies/mL measured by ddPCR. All JCV variants were wild-type and derived from patients with high antibody IV. Conclusion Stool-specific DNA extraction methods provided the highest quality of DNA, while the sensitivity of ddPCR and RT- PCR was comparable. Our findings do not support assessing stool samples for PML risk stratification in persons with MS. Further studies are needed to explore where PML-associated viral variants arise.