70 BK Polyomavirus-infected Decoy Cells in Urine Cytology Specimens of Renal Transplant Recipients

Background BK polyomavirus is one of the common post-transplant viral infections, affecting ∼15% of renal transplantation recipients (RTR), leading to graft loss in more than half of cases. Objectives Study the rate of detection of BK virus (BKV) in RTRs in Pap-stained urine cytology specimens. Methods A single center study, urine samples were collected from 99 RTR patients, with 15 Living Donors (LD) and 15 patients with chronic kidney disease (CKD) were taken as controls. And urine cytology smears were Pap stained for detection of decoy cells (DCs). Results Out of the 99 RTRs, 27 (27.3%) patients were decoy positive, 8 out of these 27 patients had uncommon DCs, and 5 out of these 27 cytology positive patients (18.5%) had biopsy proven BKV nephropathy (BKVN). Conclusion This study suggests that the finding of BKVN in 18.5% of the DC positive patients stresses the importance of screening for BK polyomavirus with Pap-stained urinary cytology in RTR. Key wards BK polyomavirus, renal transplantation, decoy cells List of abbreviations: BKV = BK virus, BKVN = BK virus nephropathy, RTR = renal transplant recipient, LD = living donor, CKD = chronic kidney disease, DC = decoy cell. Introduction pportunistic polyomaviruses infections mainly BK virus (BKV) and JC virus have become increasingly common problem among renal transplant recipients (RTR). Polyomaviruses are circular, double-stranded DNA viruses. The most important and commonest among these viruses is BKV infection, which was reported in ∼15% of RTRs in the first post-transplant year in the absence of an effective prophylaxis strategy . BKV presents with an asymptomatic gradual rise in serum creatinine with a tubulo-interstitial nephritis mimicking rejection, making a treatment dilemma. The decrease in immunesuppression that is needed to treat BKV infection is opposite to the increases in immunesuppressive drugs that are needed to treat rejection . Once the virus has reactivated, there will be an ascending infection via cell-to-cell spread . In the absence of an appropriate immunologic control, a progressive lytic infection could take place . This results in large nuclear viruscontaining inclusions in the tubular cells. Lysis of these urothelial infected cells leads to spread of the virus into the tubule lumen and then urine, as well as to the tubular interstitium and then spread to the surrounding cells. Subsequently, there will be tubular cell necrosis and cast formation . O Iraqi JMS Published by Al-Nahrain College of Medicine ISSN 1681-6579 Email: iraqijms@colmed-alnahrain.edu. iq http://www.colmed-alnahrain.edu. iq Iraqi JMS 2015; Vol.13(1) 71 Urine cytology screening for viral inclusionbearing, so called decoy cells (DCs) allows for the early identification of BKV infection, and it has a relatively high sensitivity and a negative predictive value above 95%, besides being a cost-effective non-invasive assay . Detection of DCs in the urine is one of the earliest assays, in this assay urine is Papanicolaou-stained and examined under light microscope to look for virus infected cells "decoy cells", which are epithelial cells with enlarged nuclei, and large basophilic ground-glass intranuclear viral inclusions . Thus, the objective of the present study was to evaluate the prevalence of BKV infection in RTRs based on the detection of urinary DCs in Papstained urine cytology specimens. Methods A total of 99 RTR patients who attended the (Center of Kidney Diseases and Transplantation) in the Medical City of Baghdad, were enrolled in the study. A consent letter was signed by each patient, and the study was approved by the Ethical Committee of Al-Nahrain University. Urine samples were collected from the patients, 33 of them had normal renal function, and the remaining 66 had impaired renal function. Two control groups were included in the study, 15 living donors (LD), and 15 non-transplanted patients with chronic kidney disease (CKD). Living Donors (who are apparently healthy individuals, not diabetic, not hypertensive, not receiving any medications, and their serum creatinine and creatinine clearance tests are normal). Urine (10-ml aliquots) was centrifuged in Falcon tubes at 1500 rpm for 5 min for DCs screening. The supernatant was discarded and the sediment was re-suspended in the remaining urine. For each patient; two slides were prepared; one was immediately stained with the Papanicolaou method and examined under light microscope at 40 and 100X; the other was stored unstained at -20 C for confirmation of diagnosis if required (slides preparation and staining were conducted in the Teaching Laboratories in the Medical City of Baghdad). Identification and Quantification of Decoy Cells Activation and replication of polyomaviruses was detected by identification of DCs, which are viral inclusion-bearing epithelial cells characterized by a ground-glass appearance with an enlarged nucleus, occupied by a basophilic inclusion surrounded by chromatin . Some of the DCs appear resembling the tail of a comet . For DCs quantification; a cut-off level of ≥ 10 DCs / (removed) slide, is defined as decoy positive . In addition to the quantification of common ground-glass DCs, the uncommon (clumped) variants were also looked for; as their presence reflects the pathological stages of BKVN, if the uncommon (clumped) variants are more than 25% of the total decoy cell count; then BKVN can be predicted with more than 75% probability . Statistical analysis Statistical analysis was performed with the software SPSS version 21.0, and Microsoft Excel 2013. Categorical data formulated as count and percentage. Fisher exact test was used to describe the association of these data. Numerical data were described as mean, standard deviation of mean. ANOVA was used for comparison among more than two groups. P ≤ 0.05 was considered statistically significant. Results This prospective study involved 99 RTR, 33 of them had normal renal function, and the remaining 66 had impaired renal function, 78/99 (78.79%) were males. Their mean age was 37±13years ranging between 18 and 67 years. The mean serum creatinine value in the RTRs was 2.33±1.7 mg/dl, and their mean posttransplantation period was 17.5±9.7 months ranging from 2-30 months Among these 99 RTRs, 19.2% had renal allograft rejection (biopsy-proven), five of them (5.1%) were receiving antithymocyte globulin (ATG) as anti-rejection therapy. Al-Obaidi et al, BK Polyomavirus-infected. 72 In addition, 5.1% had biopsy proven BK virus nephropathy (BKVN) (biopsy was studied in a separate laboratory), and 4.0% had ureteric stenosis (diagnosed by ultrasonography). Papanicolaou-stained urine cytology smears revealed high rate of DCs shedding among RTR as compared with both control groups; LD and CKD that were all DCs negative, table 1. On the other hand, uncommon DCs variants were present in 8 out of 99 RTR as shown in table 1 and fig. 1. The most frequent variant of DCs was the amorphous, basophilic, ground-glass–like nuclear appearance. While in the other variants (uncommon type), the nucleus appeared eosinophilic and granular, and could be surrounded by a halo, or with a finely granular without a halo (Fig. 1). Table 1. A; Decoy cells shedding, and B; Uncommon decoy cells shedding in renal transplant recipients Feature Study groups