Multiplex Real-Time PCR Assay for Simultaneous Quantification of BK Polyomavirus,JC Polyomavirus,and Adenovirus DNA

In recent years, virus-induced nephropathy caused mainly by BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), and adenovirus has emerged as a problem in renal transplant patients. In the present study, we developed a multiplex real-time PCR assay to quantify the viral load of BKPyV, JCPyV, and adenovirus simultaneously. The dynamic range covered at least 6 orders of magnitude. This system was specific and reproducible, even in the presence of large amounts of DNA of other viruses. To validate this assay, urine samples from 124 renal transplant patients and serum samples from 18 hemorrhagic cystitis patients after hematopoietic stem cell transplantation were examined. In the urine samples from renal transplant patients, BKPyV was detected in 28 patients (22.6%), JCPyV was detected in 51 patients (41.1%), and adenovirus was detected in 2 patients (1.6%). The maximum amounts of each virus detected were 2.7 × 10⁹, 8.7 × 10⁸, and 1.2 × 10² copies/ml, respectively. Decoy cells were observed in 31 patients. The quantities of both BKPyV and JCPyV DNA were greater in samples with decoy cells. Two patients whose BKPyV viral loads exceeded 10⁸ copies/ml had elevated serum creatinine levels and were diagnosed with BKPyV nephropathy based on graft biopsies. In serum samples from hemorrhagic cystitis patients, BKPyV, JCPyV, and adenovirus was detected in six, two, and three patients, respectively. Strong correlations were observed between the viral DNA copy numbers determined in the multiplex assays and those determined in single assays. Since this new assay is rapid, sensitive, and specific, it can be used for quantitative analyses of viruses in urine and serum samples after transplantation.Although immunological rejection after renal transplantation has decreased with advances in immunosuppressive therapy, virus-induced nephropathy, which was rare with conventional immunosuppressants, has become a clinical problem. BK polyomavirus (BKPyV) is a prevalent pathogen causing virus-induced nephropathy, and other viruses, including JC polyomavirus (JCPyV) and adenovirus, can cause nephropathy (1, 4, 13). These viruses commonly infect a large number of people in childhood and remain latent in the urinary tract after primary infection. Reactivation with asymptomatic viruria may occur in both immunocompetent subjects and immunocompromised patients.For the early diagnosis and treatment of BKPyV nephropathy, urine cytology is useful for screening high-risk patients (1). The virus-infected urothelial cells called “decoy cells,” identified by their typical ground glass intranuclear inclusions on cellular smears stained by the Papanicolaou method, are observed in most patients with BKPyV nephropathy. However, decoy cells are not specific for the presence of BKPyV in urine and can be found in JCPyV and adenovirus infection (1, 4, 22). Therefore, the detection of viral DNA is essential for a precise diagnosis and the identification of patients at high risk for nephropathy. In addition, hemorrhagic cystitis can be observed after hematopoietic stem cell transplantation and sometimes after renal transplantation. This involves sustained hematuria and symptoms of lower urinary tract irritability, such as dysuria with frequency and urgency. It is caused most often by adenovirus with serotypes 11, 34, and 35 in subgroup B, although BKPyV and JCPyV can also cause hemorrhagic cystitis (6, 22).Recently, real-time PCR methods have been introduced into clinical virology for the quantitative detection of viral copy numbers, and the exact determination of virus genome copy numbers provides useful information about the magnitude of the infection, which is beneficial for evaluating whether the detected virus is pathogenic, especially with latent virus infections. Previously, we developed multiplex real-time PCR systems that can quantify three viruses simultaneously (20, 21) and put the technique into clinical practice after stem cell transplantation. The use of these systems reduces significantly the time and labor required for the examination of each virus.The present study developed a multiplex real-time PCR assay for the simultaneous quantification of BKPyV, JCPyV, and adenovirus and validated it for the quantitative determination of viral loads. This method was then further validated on samples of urine and serum from posttransplant patients.