The retrospective cohort study was performed using respiratory specimens (nasal wash, tracheal aspirates, and bronchoalveolar lavages) from inpatients and outpatients at The Children’s Hospital, Aurora, CO, collected from January 1, 2008 to December 31, 2009. Approval was obtained from the Colorado Multiple Institutional Review Board. The majority of these patients had acute respiratory illnesses. The immunocompromised group consisted of patients seen on the hematology or oncology services, and included children diagnosed with hematologic or solid organ malignancies, bone marrow transplant recipients, individuals on chemotherapy, or those with a primary immunodeficiency. The controls were other inpatients or outpatients at The Children’s Hospital. Patients were excluded from the control group if they were HIV positive, solid organ transplant recipients, diagnosed with primary immunodeficiencies, or receiving immunosuppressive therapy. The majority of these patients had acute respiratory illnesses, but several in the immunocompromised group were asymptomatic.
Given the possibility of year-round viral reactivation that may occur in the immunocompromised group that may not reflect the true seasonality of the viruses, time-matched controls were selected over age-matched controls in order to match for season. 2:1 matching of controls to cases was calculated to provide a 95% confidence interval with 80% power to detect a 5% difference in prevalence from the previously reported 7% for WUPyV.3
Viral nucleic acids were extracted from the respiratory specimens using the Qiagen Viral Kit (2.0) (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The DNA primers and probes for the VP1 region of WUPyV (GenBank accession number EF444549) and KIPyV (GenBank accession number NC009238) were designed using Primer Express software 3.0 (Applied Biosystems, Foster City, CA). Basic Local Alignment Search Tool (BLAST) searching of the primer and probe sequences determined that the sequences were specific to WUPyV and KIPyV, and control PCRs with other polyomavirus VP1 sequences (JC, BK, Lymphotrophic polyomavirus (LPV), Merkel cell Virus (MCV), SV40) confirmed this analysis. Primer and probe sets for WU and KI PyV were as follows:
- WU-VP1-Forward: 5′AACCAGGAAGGTCACCAAGAAG3′
- WU-VP1-Reverse: 5′CTACCCCTCCTTTTCTGACTTGTTT3′
- WU-VP1-Probe: 5′CAACCCACAAGAGTGCAAAGCCTTCC3′
- KI-VP1-Forward: 5′CTCCCCACCAGTGTAAATCTT3′
- KI-VP1-Reverse: 5′GGAGCCTGGGACTGAAGTGTT3′
- KI-VP1-Probe: 5′CTCAGCTTCCACGCAC3′
Quantitative real time PCR was performed on the ABI Prism 7750 Sequence Detector. All real time assays used common PCR mix and cycling conditions. 12.5 μL of TaqMan Master mix (Applied Biosystems, Foster City, CA), 2.5 μL of 900 nM primer, 2.5 μL of 250 nM of the corresponding probe, and 50 ng of sample nucleic acid extract in a final reaction volume of 25 μL, was cycled under the following parameters: incubation of 2 min at 50 °C, followed by 10 min at 95 °C, 45 cycles of denaturation at 95 °C for 15 s, and extension at 60 °C for 1 min. Results were expressed as cycle threshold (Ct).
Plasmid DNA standards for each viral target were included in each PCR assay at concentrations of 10, 100, 1000, 10,000 and 100,000 copies per 50 μL reaction to verify assay sensitivity. Results were converted to copy number by plotting the Ct value on a standard curve using serial dilutions of a control plasmid of known concentration. Viral copy numbers were expressed both as copies per mL of sample and copies per cell. Viral copy numbers were normalized to human cells in the specimen using the C-Reactive Protein (CRP) gene copy number. The number of cells present in the sample were calculated from the value of 106 CRP copies per 500 ng of DNA, determined using the standard curve method described above. Viral copy numbers per cell were obtained by dividing viral copy number per μL by the number of cells per μL. Viral copy numbers per volume correct for the sample volume required to extract 50 ng of nucleic acid.
The assay was tested by using nasopharyngeal specimens found to contain influenza A virus, influenza B virus, respiratory syncytial virus (RSV), parainfluenza, adenovirus and human metapneumovirus (hMPV) as controls. In addition, each assay was tested using human genomic DNA, as well as WUPyV and KIPyV plasmids. There was no cross-amplification detected with these specimens, indicating the PCR assay’s high specificity. The primers, probes, and assay characteristics for CRP have been described elsewhere.6
The majority of specimens were tested for the presence of other respiratory viruses using the ID Tag Respiratory Viral Panel (Luminex Molecular Diagnostics, Toronto, Canada) for the following viruses: (RSV) A, B, influenza A and B, parainfluenza 1, 2, 3, 4 (hMPV), adenovirus, coronaviruses 229E, OC43, NL63, HKU1, and enterovirus/rhinovirus. The specimens that tested positive for WUPyV and KIPyV that had previously only been tested with direct fluorescent antibody (DFA) and viral culture were subsequently tested with the entire respiratory viral panel. The panel results were not quantified for copy number, but given only as present (positive) or not present (negative).