Prevalence of WU and KI polyomaviruses in plasma, urine, and respiratory samples from renal transplant patients

Test Specimens

One Hundred and ninety five blood samples from 195 patients (82 women, 113 men; median age: 45.7 years; range: 7–68.8 years) were collected at different times after renal transplantation (median: 1188 days, range: 3–7108). For control measurements, 200 blood samples from 200 healthy blood donors were taken (75 men, 125 women, median age: 39 years, range: 10–74 years). Fifty urine samples from 50 transplant patients were also collected after transplantation (range: 5–6230 days; median: 141 days). Thirty-six urine specimens from healthy blood donors were used as controls. Ninety upper respiratory tract specimens using throat swabs from 90 renal transplant individuals were obtained 18–6230 days after the transplantation (median: 1177 days).

Nucleic acids from 200 µl plasma, centrifuged for 10 min at 180 × g at 4°C, 200 µl urine specimen and throat swab sample washed in 200 µl buffer were isolated using High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Nucleic acid was eluted in 50 µl and stored at −20°C until use.

The Regional and Institutional Ethics Committee of University of Debrecen approved all of the studies. All patients gave their written informed consent.

Qualitative and Quantitative Detection of KIPyV and WUPyV DNA

To detect WUPyV and KIPyV DNA, the first round of WUKI nested-PCR was carried out with WUKI_OS and WUKI_OAS primers as described previously [Sharp et al., 2009] in a final volume of 20 µl containing 5 µl DNA solution, GenAmp Fast PCR Master Mix (Applied Biosystems, Foster City, CA) and 10–10 pmol of each primer. For the second round, 4 µl of the PCR product from the first round was amplified in 20 µl final volume using GenAmp Fast PCR Master Mix and 10–10 pmol WUKI_IS and WUKI_IAS primers [Sharp et al., 2009]. The annealing temperature was 60°C in both rounds. Plasmids containing the genome of WUPyV and KIPyV were used as positive controls [Gaynor et al., 2007; Lindau et al., 2009]. The sensitivity of this nested-PCR was <100 genome equivalent/ml (GEq/ml). Since this PCR method amplifies both WUPyV and KIPyV DNA and does not differentiate between them, the PCR products were sequenced by using the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). At the same time, real-time PCR for WUPyV and also for KIPyV was performed with primers and probes described previously [Lindau et al., 2009] with 5 µl DNA to confirm the result of the nested-PCR and to quantify the viral loads in the samples.

Chi-squared and Fisher's exact test was used to assess the difference in frequency for categorical variables. Mann–Whitney U-test was applied for continuous variables. A difference was considered significant if P-value was less then 0.05.

PCR Prevalence of WUPyV and KIPyV in Plasma Samples

Seven (3.6%) of 195 plasma samples from transplant patients and none from 200 healthy blood donors were positive by WUKI PCR (P < 0.01; Table I). Sequencing of the PCR products revealed that two samples were positive for KIPyV and five for WUPyV DNA. The level of DNA load in six plasma samples was less then 250 GEq/ml urine, below the limit of detection, and 2.5 × 10² KIPyV GEq/ml in one specimen. Significant difference was found between polyoma-positive and negative samples regarding the time after renal transplantation (P = 0.001; Table II).

PCR Prevalence of WUPyV and KIPyV in Urine Samples

Seven (14%) urine specimens from transplanted patients and none from 36 healthy blood donors were positive for WUKI by PCR (P < 0.05; Table I.). One sample was KIPyV DNA positive (viral load was <250 GEq/ml plasma) and six samples were WUPyV DNA positive by sequencing. The viral loads of four samples were <250 GEq/ml, and two samples had 5 × 10² and 1.1 × 10³ GEq/ml plasma. PCR positive samples were collected significantly earlier after transplantation then the negative samples (P = 0.001; Table II). In the case of two patients whose urine samples were WUPyV DNA positive (28.6%), WUPyV viremia was also detected.

Prevalence of WUPyV and KIPyV by PCR in Respiratory Samples

Nine (10%) of 90 respiratory samples were WUKI PCR positive (Table I). Six samples were KIPyV DNA positive (66.7%) with viral loads ranging from 2.8 × 10² to 3.7 × 10⁵ GEq/ml (median: 4.2 × 10⁴; in two samples the viral load was below the limit of detection). Only one out of the three WUPyV positive samples had detectable viral load of 6.3 × 10² GEq/ml. Statistical analysis revealed that the PCR positive samples were collected significantly earlier after transplantation then the PCR negative samples (P = 0.002; Table II). The plasma sample of one patient with a KIPyV positive respiratory specimen was positive for WUPyV DNA, the plasma samples of the others were PCR negative. All of the patients with a positive respiratory specimen had acute upper respiratory tract infection, but none of these samples were tested for any respiratory virus; a significantly higher frequency compared with the PCR negative patients (9/9 vs. 47/81; P = 0.01).DISCUSSION

This study revealed that WUPyV and KIPyV can be detected in blood, urine, and respiratory samples from renal transplant patients, but these viruses were not found in blood and urine specimens from healthy blood donors.

Viremia was detected in 3.6% of the renal transplant patients, mostly early after transplantation, but not in healthy blood donors. Other studies have found only WUPyV in blood samples [Bialasiewicz et al., 2009; Miller et al., 2009; Neske et al., 2009], but apart from WUPyV (2.6%), KIPyV (1%) was detected in the current study in renal transplant patients. The viral loads were very low, ≤250 GEq/ml plasma. Previously, WUPyV, KIPyV were not found in urine samples from immunocompromised, renal transplant patients, immunocompetent patients, and pregnant women [Gaynor et al., 2007; Bialasiewicz et al., 2009; Bofill-Mas et al., 2010]. In this study these viruses were not detected in urine specimens from healthy blood donors, but 14% of the samples from renal transplant patients were positive for viral DNA. A higher prevalence of WUPyV was observed compared with KIPyV (6/7 vs. 1/7). The viruses appeared early after renal transplantation, and all of the positive samples were collected within 2 months after the transplantation. The viral loads in these urine samples were low (≤1.1 × 103 GEq/ml), but in two patients WUPyV viremia was also found at the same time. A different DNA isolation method in which samples were not stored and DNA was isolated immediately after the collection of the urine samples, primers, and PCR conditions may be the reasons why these viruses were found while other investigators did not detect these viruses in urine samples [Gaynor et al., 2007; Bialasiewicz et al., 2009; Bofill-Mas et al., 2010].

In immunocompetent individuals a slightly higher prevalence of WUPyV DNA was observed in the respiratory samples [Dalianis et al., 2009] and also the seroprevalence of WUPyV was found to be greater [Nguyen et al., 2009; Neske et al., 2010] compared with KIPyV. In the throat swab samples from renal transplant patients a higher prevalence of KIPyV (6/9; 6.7%) then WUPyV (3/9; 3.3%) was observed. This is in accordance with the results of Mourez et al. [2009] who found a higher frequency of KIPyV in respiratory samples from immunocompromised patients. A significant difference was found between the PCR positive and the negative group of the patients regarding the date of samples collection. Viral DNA was detected mostly early after renal transplantation.

The results of previous studies suggest that primary infection with WU and KI viruses occurs during childhood with subclinical or mild illness [Bialasiewicz et al., 2007; Gaynor et al., 2007; Abedi Kiasari et al., 2008; Neske et al., 2010]. Transmission can be fecal–oral and/or via the respiratory route [Dalianis et al., 2009]. Presumably, in a manner similar to BKV and JCV, it may establish lifelong persistence [Jiang et al., 2009]. This study revealed that viruria with WUPyV and KIPyV can occur, so urine can also be a source of infection. The higher prevalence of WUPyV and KIPyV in respiratory samples from immunocompromised patients, and the findings that these viruses are present in blood and urine specimens from renal transplant patients, but not in healthy blood donors, suggest that similar to BKV and JCV, WUPyV and KIPyV might cause significant disease primarily in immunocompromised individuals. At the same time, because of the high rates of coinfections with other respiratory viruses, the pathological role and the clinical consequences of the KI and WU respiratory tract infections are not clean. Based on the knowledge of other human pathogen polyomaviruses it is hypothesized that immunosuppression due to transplantation may result in reactivation of these viruses, or may establish greater susceptibility to KIPyV and WUPyV [Jiang et al., 2009]. Although viremia and viruria were found in the current study in renal transplant patients, the viral loads were low. In the case of BKV the level of viruria correlates with the degree of immunosuppression, and the higher viral loads in urine and blood samples can result in more severe clinical consequences [Ahsan and Shah, 2006]. Further follow up studies of renal transplant patients may help to clarify whether the presence of WUPyV and KIPyV in urine and blood samples can result in severe disease as observed with BKV.