CSF specimens and clinical data
The study was approved by the medical research ethics committee for the use of human subjects at the National Institute of Infectious Diseases (NIID), Tokyo, Japan (approval numbers 667 and 964). It was performed in accordance with the ethical standards of the Declaration of Helsinki. Written informed consent was obtained from patients or their families.
In Japan, real-time PCR testing of CSF specimens for JCV DNA is routinely performed at the Department of Virology 1, NIID [9, 20,21,22,23,24,25,26]. For this study, CSF specimens were collected by lumbar puncture from 21 individuals. Of these, one patient had previously been diagnosed with PML. The JCV-positive CSF obtained during follow-up of this patient was diluted with Dulbecco’s phosphate-buffered saline (DPBS, sterile-filtered, calcium- and magnesium-free, Thermo Fisher Scientific Inc., Waltham, MA) and used as a standard virus suspension to compare the performance of our PCR assay. The remaining 20 patients had suspected PML based on neurological symptoms and/or brain MRI findings. The initial CSF specimens were subjected to real-time PCR testing to detect JCV DNA with and without undergoing ultrafiltration.
All CSF samples were frozen at the source hospitals without centrifugation and were transported to the NIID in solid carbon dioxide, at which point they were stored at − 80 °C until further analyses. Anonymous clinical data were obtained from physicians, using standardized questionnaires. The data were analyzed with respect to age, gender, underlying disease, neurologic symptoms, and brain MRI findings.
CSF ultrafiltration
JCV particles in fluid samples were concentrated using an Amicon Ultra-4 10 K centrifugal filter device with a 10 kDa molecular weight cut-off membrane (EMD Millipore Corporation. Billerica, MA; hereafter called filter device). Centrifugation was executed with an Eppendorf 5804R centrifuge and an A-4-44 swing rotor (Eppendorf, Hamburg, Germany). The filter device, contained in a 15-mL centrifuge tube (BD Biosciences, Franklin Lakes, NJ), was spun in a centrifuge bucket sealed with an aerosol-tight cap (Eppendorf) for the use of clinical specimens that may contain potentially infectious agents. In addition, all centrifuge equipment (e.g., swing rotor, buckets, and sealing caps) were routinely irradiated with ultraviolet light to prevent DNA contamination before and after examination. The handling procedures are summarized in Fig. 1.
Frozen specimens were thawed at 37 °C in a block incubator (DTU-Neo; Taitec, Saitama, Japan) until the ice had just melted, before being mixed and temporarily stored in a refrigerator. To wash glycerol from the ultrafiltration membranes, 4 mL of DPBS was added to the filter device and spun at 4000×g for 4 min at room temperature. The filtrate in the centrifuge tube and any residual liquid in the filter device were discarded, and these wash steps were repeated to a total of three times. Next, a 4-mL CSF sample was added to the filter device in a fresh 15-mL centrifuge tube and was centrifuged at 4000×g for 8–10 min at 25 °C until the concentrate volume was slightly less than 500 μL. The concentrate was stirred by flushing with a pipette at least five times inside the filter device, and its volume was accurately adjusted to 500 μL with DPBS. The concentrate was mixed with 500 μL of lysis buffer containing guanidine hydrochloride (Buffer AL; Qiagen, Venlo, Netherlands) and subjected to nucleic acid extraction and real-time PCR analysis.
DNA extraction
Total DNAs were extracted from CSF samples using a QIAamp MinElute Virus Spin Kit (Qiagen), according to the manufacturer’s protocol with slight modification. This kit is designed for DNA/RNA extraction with a starting sample volume of 200 μL. To obtain more concentrated extracts from larger samples, we used 500 μL of original or ultrafiltrated CSF samples for DNA extraction and used the manufacturer’s protocol for a similar extraction kit (QIAamp MinElute Virus Vacuum Handbook 3rd edition; Qiagen). Briefly, the CSF sample was mixed with 500 μL of Buffer AL, 75 μL of Qiagen Protease (1.07 Anson units/mL; Qiagen), and 5.6 μL of carrier RNA (1 μg/μL; Qiagen). After incubation at 56 °C for 15 min in the block incubator, 600 μL of 99.5% ethanol was added to the lysate, and the resulting mixture was passed through the MinElute column for centrifugation at 6000×g for 1 min, three times. The MinElute column was washed, 30 μL of distilled water (UltraPure DNase/RNase-Free Distilled Water; Thermo Fisher Scientific Inc.) was applied, and 25 μL of DNA extract was collected. DNA samples were then subjected to real-time PCR assay.
Real-time PCR assay
Quantitative real-time PCR assay targeting the JCV large T gene was carried out, as described in the earlier reports [9, 20,21,22,23,24,25,26]. To confirm the detection of JCV genomic DNA, real-time PCR for the JCV viral protein 1 (VP1) gene was conducted [9]. In addition, sample contamination with standard DNA plasmid was monitored by real-time PCR that detected standard DNA but not the JCV genome [9]. These PCR assays were capable of detecting at least four copies of the target DNA per reaction [9].
Statistics
Differences in the copy numbers of JCV DNA in CSF specimens were compared by parametric analyses using the paired Student’s t-test. All P-values less than 0.05 were considered statistically significant.