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

Abstract

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 ؋ 10 9 , 8.7 ؋ 10 8 , and 1.2 ؋ 10 2 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 10 8 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 hematopo...

Choreito Formula for BK Virus–associated Hemorrhagic Cystitis after Allogeneic Hematopoietic Stem Cell Transplantation

“…This qualitative PCR could not detect BKV in patients without HC. After January 2010, viruses were monitored by multiplex RT-PCR for quantification of DNA from BKV, polyomavirus JC, and AdV, as described previously [16]. In April 2010, BKV RT-PCR was used to screen all 30 hospitalized children with various hematooncological diseases who had neither HC-related symptoms nor abnormal urinalysis.…”

Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants

“…Consequently, identifying JCV genotypic variants as non-virulent, or virulent, provides value in determining which patients may be at greater risk of developing PML. A previous qPCR assay described as multiplex distinguishes JCV from other DNA viruses (14), but does not distinguish JCV variants as detailed in the following.…”

Significance of decoy cells for indicating viral cytopathic effect in urine cytology

“…Dear Editor, Decoy cells are well known in urine, with homogeneous, hyperchromatic nuclei mimicking cancer cells, but which are associated with BK polyoma virus (BKPyV) . However, cells with similar cytopathic changes have been observed with other viral infections . Further identification by immunostaining or in situ hybridisation is recommended to avoid misdiagnosis.…”

“…Electron microscopy is also useful to demonstrate viral particles specific to each virus in the infected cells . In addition, real‐time polymerase chain reaction for the simultaneous quantification of several viruses, including BKPyV, can be used for the analysis of urine and serum . Nevertheless, urinary cytology with Papanicolaou stain is still an efficient and cost‐effective screening method for the monitoring of patients at risk for viral infection of the kidneys and urinary tract …”

“…It has been shown recently that decoy cells are not specific for the presence of BKPyV in urine and can be found in JCPyV and adenovirus infection . Adenoviral inclusions can be mistaken for those of cytomegalovirus, herpes simplex virus or PyV, including BKPyV and JCPyV .…”