Journal of Global Pharma Technology

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1 Journal of Global Pharma Technology Available Online at ISSN: RESEARCH ARTICLE The Possible Role of JC Polyomavirus after Kidney Transplantation Asmaa B Al-Obaidi 1, Haidar A Shamran 2*, Alaa Ghani Hussein 3, Mervit Bassim Jasim 1, Ali JH Al-Saedi 4, Haider Faisel Ghazi 1 1. Department of Microbiology, College of Medicine, University of Al-Nahrain, Baghdad/Iraq. 2. Medical Research Unit, School of Medicine, University of AL-Nahrain- Baghdad/Iraq. 3. Department of Pathology, College of Medicine, University of AL-Nahrain, Baghdad/Iraq. 4. Center of Kidney Diseases and Transplantation (Ministry of Health). Department of Medicine, College of Medicine, University of Baghdad, Baghdad/Iraq. *Corresponding Author: Haidarahmad2000@yahoo.com Abstract Background John Cunningham virus (JCV) is one of the highly sero-prevalent viruses worldwide, and after primary infection it remains latent and then reactivates in immunocompromised patients, high JCV viruria seen in renal transplant recipients (RTR), some of them progress to polyomavirus associated nephropathy (PVAN). The objectives of this study are to determine JCV viruria and viremia in RTR and correlate them with urine cytology results and patients' renal function. Methods a prospective study conducted from October 2015 to to March Seventy-one (71) RTR, and 71 apparently healthy age and sex-matched normal donors as controls were enrolled in this study. Urine samples taken from all RTR cases and control, while plasma was taken from only 50 RTRs and 50 controls. Urine cytology smears were prepared and Pap-stained for decoy cells (DC) screening. Viral DNA was extracted from 1 ml urine, and from 200µl for plasma samples, and then the viral DNA was detected and measured by a real time-pcr instrument, using q RT-PCR kits for JC virus. Results Thirty one (43.70 %) of RTR and 10% of the controls were JC viruria positive, the mean of JCV viruria was 6.8 x10 4, and 1.04x10 3 copies/ml for RTRs and controls respectively (p<0.001), and 19/33 (57.58) of RTRs who had abnormal creatinine clearance were JC viruria positive (p=0.025). All 50 RTRs and the 20 controls had negative JC viremia. The rate of occurrence of DC in urine cytology was 28.17% for RTRs while none of the control subjects had DC in urine, but no correlation between JC viruria and the rate of decoy cell shedding in RTRs. Conclusion High prevalence of JCV viruria in RTRs, and associated with impairment of their renal function, which might suggest a pathological role of this virus have impact on renal function. Keywords: JC Polyomavirus, Kidney transplantation, Real-time PCR. Introduction During the last decade, the human polyomaviruses have become important clinical entities, coincident with the development and use of more potent immunosuppressive agents. Polyomavirusassociated nephropathy (PVAN) is one of the important causes of graft dysfunction with a high rate of graft loss (1). BK virus (BKV)- associated nephropathy is a well-defined entity. In addition to BKV, JC virus (JCV) may also be involved in PVAN (2). The JC polyomavirus is a small non-enveloped, circular, and double-stranded DNA genome. Belonging to the family Polyomaviridae (3).The virus infects the majority of humans. Approximately 60 80% of adults in the United States have detectable antibodies against JC virus (3,4). Primary infection with JC virus occurs mainly during adolescence and early adulthood, when it presents as a benign and asymptomatic infection. Thereafter, JC virus establishes lifelong latency in the kidneys, central nervous system, and hematopoietic progenitor cells (5,6), JCV is the causative agent of PML which is often fatal disease and is characterized by lytic infection of the glial cells (7). JCV has also been linked to nephropathy in the context of renal , JGPT. All Rights Reserved 580

2 transplantation, although much less frequently than BK virus (8,9).Studies showed that JC viral load was markedly increased in kidney transplant recipients, in liver transplants JC virus infection was reported in about 1.7%, and 22.7 % of recipients excreted the virus (10,11). Infection by JCV was observed in RTR as both nephropathy and/or PML. RTRs have the highest risk of developing JCV- associated nephropathy as immune competent individuals, but in RTRs it is more common to observe a higher level of polyomavirus replication, as demonstrated by urinary shedding of decoy cells (15,16). Few cases of nephritis have been attributed to JCV as compared to BKV, and also limited information is available on JCV replication, viruria and decoy cells shedding in RTRs, and its effect on renal function and survival (3,16). Kusne et al in 2012, showed that lower mean creatinine clearance (Cr Cl) values were significantly associated with JCV shedding in both kidney and liver recipients(17). In Iraq, to the best of our Knowledge, only one study was conducted on JCV, which detected this virus in colorectal cancer tissue by real time PCR and found the virus in 30% of colorectal adenocarcinoma (18). Few studies investigated viral infections in Iraqi renal transplants including BK virus (19), human cytomegalovirus (20), Epstein Barr virus (21) and Human herpes virus-6 (22). This study aimed to investigate the rate of occurrence of JC polyomavirus in a sample of Iraqi RTRs, and to correlate it with patients' creatinine clearance and the type of immunosuppressive regimens. Patients and Methods A prospective study conducted from October 2015 to March 2016, seventy-one (71) RTRs were collected from the (Center of Kidney Diseases and Transplantation) in the Medical City of Baghdad, and 71 age and sex-matched normal donors enrolled in this study as controls. A consent letter obtained from all patients and controls enrolled in the study. This study approved by the ethical committee of the College of Medicine-Al-Nahrain University. Clinical parameters (immunosuppressive regimens, acute rejection episodes, transplant function and late complications) obtained from patient's medical records. Two main Standard immunosuppressive regimens were mainly compared to other organ transplant recipients. Polyomavirus-associated nephropathy (PVAN) associated with graft dysfunction and graft loss has been of particular importance since 1990S (12,13). The risk factors for JCV nephropathy are controversial and might involve multiple determinants, but immunosuppressant has been accepted as the most important key factor (13,14). Low level JCV is common in followed in RTRs; either the cyclosporine (CYC), my cophenolate (MMF), and prednisolone, or the regimen that included tacrolimus (TAC) instead of CYC, in addition to MMF and prednisolone. And induction with monoclonal anti-cd25 antibodies (Basilixibam/ Daclizumab) Treatment with oral CYC was started before surgery (10 mg/kg/d) to obtain therapeutic CYC blood levels, and then was adjusted, based on a target level of ng/ml in the first four weeks, and then ng/ml thereafter. The maintenance dose of MMF was g/d. Methylprednisolone 5.0 mg/kg/d was administered on three consecutive days from the day of RT. While oral prednisolone was started on the first day after operation at 0.5 mg/kg/d and reduced gradually till mg/d. For those patients who were on TAC regimen; the starting dose was 0.05 mg/kg at induction, then mg/kg according to the blood level which should be 6-12 n g /mlin the first 3 months and then 4-8 ng/ml maintenance immunosuppressant. From all RTRs and controls, 10 ml urine samples collected and divided into two halves, 5ml were preserved in deep freeze for viral DNA extraction, and the second 5ml were preserved in 95% ethanol 1:1 for Pap-stain urine cytology smear. A 2.5 ml whole blood in EDTA tubes obtained from 50 RTRs of these 71 patients and from the controls. Urine Cytology Urine (10-ml aliquots) centrifuged in white cap tubes at 1500 rpm for 5 min for decoy cell screening. The supernatant was discarded and the sediment was re-suspended in the remaining urine. For each patient two slide was prepared, for identification of decoy cells, the slides stained with the Papanicolaou method and examined under light microscope at 10X and 40sX.Decoy cell (DC) are viral inclusion-bearing epithelial cells characterized by a ground-glass appearance with an , JGPT. All Rights Reserved 581

3 enlarged nucleus, occupied by a basophilic inclusion surrounded by chromatin (23,24). S some of the DCs typically appear like the tail of a comet(25). For DCs quantification a severance level of 10 DCs/slide, defined as decoy positive(26). Viral DNA Extraction For viral DNA extraction from the plasma and urine samples Geneius Micro g DNA Extraction kit (Gene aid, England) was used. Viral DNA extraction method based on the silica-membrane column separation. Quantification of JC Viral Load Gene Proof PCR kit ISIN Version kit (England) is a Real-Time test for the quantitative detection of JCV in the biological materials was used. Thirty (30) µl of Master Mix was added into PCR tubes and then 10 µl of the (sample DNA, positive or negative controls, or standards (four: standards JCV copies/ml) added into the individual PCR tubes. The final reaction mix volume was 40 µl. Real times PCR instrument used in this work was STRATAGENE M x Pro QPCR (Agilent Technologies, USA). The thermal protocol for Gene proof PCR kit is composed of a two hold steps at 37 C for 2 min, and then at 95 C for 10 min, and 45 amplification cycles of 95 C for 5 sec, 60 C for 40 sec and 72 Cfor 20 sec. The real-time data is collected at the third step of the amplification cycle. According to the manufacturer instructions, for JCV positive samples, a JCV DNA copy was calculated according to the following formula: SC- Sample Concentration (copy/µl), EV- Elution Volume (µl), IV- Isolation Volume (ml) Statistical Analysis The Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA), Version 20 was used for statistical analysis. Categorical data formulated as count and percentage. Chi-square test used to describe the association of these data. Alternatively, Fisher exact test was used if there is 25% of cells less than expected count. Numerical data were described as mean and standard deviation. Independent sample t-test used for comparison between two groups. Relative risk (RR) is the ratio of the probability of pathogen occurring in an exposed group to the probability of the event occurring in a comparison, non-exposed group. The lower level of accepted statistical significant difference is Result Among the 71 RTR; 61 (85.90%) were males, and 10 (14.10%) were females; their mean age was 38.54±8.01 years, ranging between 18 and 77 years. The mean serum creatinine value of the RTR was 1.21±0.35, and the mean of their creatinine clearance was 83.39±35.11.While their mean posttransplantation period (PTP) was 6.9±4.37 months, ranging between 1-16 months. All the RTRs received their allograft from living donors, and out of the 71 RTRs; 22 (30.99%) received their allograft kidney from living related donors, while the remaining 49 (69.01) received their kidney from living unrelated donors. Papanicolaou-stained urine cytology smears showed that 20/71 (28.17) of RTRs had positive DC; while all of the controls were decoy negative which was statistically significant (p=0.005). Thirty one (31) out of 71 (43.7%) RTRs, and (10%) of the controls had positive viruria, which was significantly higher in RTRs, (p=0.007). The mean of JCV viruria was 6.8 x10 4, and 1.04x10 3 copies/ml for RTRs and controls respectively (p<0.001). QRT-PCR run conducted on the plasma of 50 out of these 71 RTRs and controls. However, none of these RTRs and controls had detectable JCV viremia , JGPT. All Rights Reserved 582

4 Figure 1: Pap-stained urine cytology smears demonstrate: (A and B) Common ground glass polyomavirus-infected (decoy cells (DC)). (C) Common comet-shaped DC. (D) Uncommon clumped variant DC. Magnification power for A (100X), B, C,D (40X) Table 1: Correlation JC viruria and RTRs' descriptive data Viruria positivity Total P value RR CI Present Absent Age groups <40 years 15 (42.86) 20 (57.14) >=40 years 16 (44.44) 20 (55.56) 36 Gender type Female 4 (44.44) 5 (55.56) Male 27 (43.55) 35 (56.45) 62 Donor relation Un related 19 (38.78) 30 (61.22) Related 12 (54.55) 10 (45.45) 22 PTP < 6 months 16 (55.17) 13 (44.83) >= 6 months 15 (35.71) 27 (64.29) 42 Serum Creatinine Abnormal 15 (60) 10 (40) Normal 16 (34.78) 30 (65.22) 46 Creatinine clearance Abnormal 19 (57.58) 14 (42.42) Normal 12 (31.58) 26 (68.42) 38 DC Absent 20 (39.22) 31 (60.78) Present 11 (55) 9 (45) 20 IS regimen CYC 21 (50) 21 (50) TAC 10 (34.48) 19 (65.52) 29 Shifting from No 31 (49.21) 32 (50.79) > 1.46-> CYC to TAC Yes 0 (0) 8 (100) 8 PTP: post-transplantation period, DC: decoy cells, IS: immunosuppressant, CYC: cyclosporine, TAC: tacrolimus Discussion In this study JCV was investigated in both urine and plasma of RTRs using QRT-PCR, and all the RTRs had negative JC viremia in plasma, but 31 out 71 (43.66 %) of them had positive JCV viruria (JCV DNA in urine) the frequency was higher in comparison to other studies, ranging from 13.7 to 36.8% (27-29). And these results were significantly higher among RTRs as compared to apparently healthy normal donors controls (P=0.007). These results are supported by other studies that showed high JCV viruria and very low JCV viremia among RTRs (10,30-34). JCV was identified in kidney biopsy tissue and/or urine in a range of 3.4% and 46% of RTRs, while JC viremia ranged from 0% and 25 % (10). One possibility is that the extent of tissue involvement by JCV is less than that in BKV nephropathy. This would correlate with the low level and focal viral cytopathic changes in the renal biopsies with JCV nephropathy. Accordingly, the plasma JC viral loads in these patients might be detectable but low, or may remain below the threshold of detection (27).This hypothesis would be in line with the overall better outcome of JCV nephropathy, and JC viral load thresholds that could be used for the diagnosis of JC nephropathy in the clinical setting remain to be determined (27) , JGPT. All Rights Reserved 583

5 Another possible explanation is that the damage to the kidney is aprerequisite for access of virus to the blood stream, and JCV is slowly pathogenic in the kidney and therefore does not access the blood streamas rapid as BKV (35). The fact that the JCV has the potential to cause renal disease is that less common than BKV, and JCV mostly has been documented in isolated cases (10, 36, 37). Specifically, JCV-mediated nephropathy has been reported by [Kazory et al] (8), and [Wen et al ] (2) in RTRs. Recently, a study showed that infection of primary human renal tubule epithelial cells with JCV and BKV results in divergent innate immune responses that control JCV but fail to control BKV. It is the first study that directly compares JCV and BKV infection in vitro in the same cell type they naturally infect, and the significant differences that have been uncovered could in part explain the distinct disease outcomes (38). The study of Drachenberg et al., (2007) had been investigated one fifth of RTRs with JCV shedding, developed biopsy-proven JCV nephropathy (i.e., viral cytopathic changes, nuclear SV40 in epithelial cells, and also with associated tissue reaction) (27). In the current study, 60% of those RTRs with impaired renal function had positive JC viruria which is statistically significant (P=0.048), that agree with other studies which suggested a role of JCV in allograft nephropathy in RTRs just like BKV (3,17,27,39,40). In addition, about 58% of those who had abnormal creatinine clearance (Cr Cl) also had positive JC viruria, which is significantly higher than those who had negative JC viruria. A finding also reported by the study of (Kusne et al.) who showed that high JCV shedding was significantly associated with lower mean CrCl values in both kidney and liver allograft recipients (P <.001) (17). Analysis of Kusne et al., suggested that JCV might contribute to renal dysfunction in some patients. This finding is consistent with reports that JCV can be linked to some cases of (polyomavirus-associated nephropathy) PVAN in RTRs (17,27,41). So, that the current studies support Kusne study and can suggest a need for deeper investigation to those transplanted patients who develop unexplained slowly progressive reduction in their creatinine clearance values. This study found that only 11 out of 31 (35.48%) positive JC viruria patients had positive decoy cells (DC) in Pap-stained urine cytology, which was statistically not significant. Whereas the prevalence ofuncommon DC was 7/71 (9.86%). Which is consistent with study by Drachenberg et al., (2007) (27) which showed low incidence of DC inrtrs, that was 13.8% of 103 patients, and it was not significantly associated with JC viruria. Based on the morphologic features of DCs alone, one cannot always distinguish among different viral infections. DCs might result from infection with BKV, JCV, and less commonly, adenoviruses (42-44). Decoy cell shedding means there is a risk of PVAN (27) but mainly due to BKV which most of studies showed significant correlation with DC shedding (45,46). Even a study in Iraq in the same center showed highly significant association between BK viremia and DC even uncommon DC (20). Which might give a clue to the non-significant association between JC viruria and decoy cell in the current study, that's to say, BKV is suspected to be the cause of these DCs in the urine of RTRs in the current study. Immunosuppressive drugs were considered as risk factor for reactivation of JCV in RTRs as documented by different studies (27, 34). Although in this study statistically, there was no significant correlation between JCV and type of IS regimen, 21 out of 31 (67.7%) positive JC viruria were on cyclosporine (CYC) regimen, and that agrees with other studies (34,47,48), that showed CYC is a risk factor for JCV reactivation. In addition, the current study found that all of the patients who were shifted from CYC to TAC regimens had negative viruria which is a significant result (P=0.007). Suggesting a role of CYC in the development JCV viruria. Another study showed there were no cases of JC-PVAN among the patients receiving tacrolim us as a part of their immunosuppressive therapy(49). While other studies have failed to find a correlation between the frequency of JCV viruria and the use of immunosuppressive drugs (10,50) , JGPT. All Rights Reserved 584

6 However, in this study, CYC use was an important independent predictor of JCV infection. The explanation that CYC has greater peak inhibition of calcineurin activity in vivo (51), which could lead to a greater effect on T-cell function and T-cell immunity have important role in protection against JCV infection. Moreover, the greater degree of immunosuppressant generated by a CYCbased triple immunosuppressive regime than a similar FK506- based regimen may be involved in this disparity (34). Finally, JC-PVAN is a unique clinical entity that should be differentiated from BK-PVAN. This requires viral typing methods that are not widely available, and this should account for an underestimation of its incidence in RTRs. However, the slow and non-aggressive clinical course of thedisease and the References 1. Hirsch HH, Knowles W, Dickenmann M et al (2002) Prospective Study of Polyomavirus Type BK Replication and Nephropathy in Renal-Transplant Recipients. N Engl, J, Med, Aug, 15;347(7): Wen MC, Wang CL, Wang M et al (2004) Association of JC Virus with Tubulointerstitial Nephritis in a Renal Allograft Recipient. J Med, Virol,72(4): Delbue S, Ferraresso M, Ghio L et al (2013) A Review on JC Virus Infection in Kidney Transplant Recipients. 4. Padgett BL, Walker DL (1973) Prevalence of Antibodies in Human Sera against JC Virus, an Isolate from a Case of Progressive Multifocal Leukoencephalopathy,127(4). 5. Arthur RR SK (1989) Occurrence and significance of papovaviruses BK and JC in the urine. Prog Med Virol,36: Schneider EM DK (1993) High frequency of polyomavirus infection in lymphoid cell preparations after allogeneic bone marrow transplantation. Transpl Proc,25: Delbue S, Ferraresso M. JC Polyomavirus Infections in Transplant Patients. 2012;2(3): Kazory a, Ducloux D (2003) Renal transplantation and polyomavirus favorable clinical outcome should be considered once this form of PVAN is diagnosed. Thus, monitoring of JCV infection, especially during the first 24 months posttransplantation, is recommended and the development of new, more sensitive technologies will be advantageous. One limitation of the present study was lack of confirmation of the detection by kidney biopsy because of unapproved protocol biopsy in the center of kidney diseases and transplantation in Baghdad. Acknowledgement Authors would like to acknowledge the Center of Kidney Diseases and Transplantation in The Medical City of Baghdad. infection: recent clinical facts and controversies. Transpl Infect Dis,5(2): Kantarci G, Eren Z, Demiraǧ A et al (2011) JC virus-associated nephropathy in a renal transplant recipient and comparative analysis of previous cases. Transpl Infect Dis,13(1): Randhawa P, Uhrmacher J, Pasculle W et al (2005) A comparative study of BK and JC virus infections in organ transplant recipients. J Med Virol,77(2): Razonable RR, Brown RA, Humar A et al (2005) Brief Report A Longitudinal Molecular Surveillance Study of Human Polyomavirus Viremia in Heart, Kidney, Liver, and Pancreas Transplant Patients,192: Hirsch HH, Steiger J (2003) Polyomavirus BK. Lancet Infect Dis, 3(10): Hirsch HH, Polyomavirus BK (2002) nephropathy: a (re-)emerging complication in renal transplantation. Am J Transplant,2(1): Comoli P, Binggeli S, Ginevri F, Hirsch HH (2006) Polyomavirus-associated nephropathy: Update on BK virus-specific immunity. Transpl Infect Dis,8(2): Drachenberg CB, Papadimitriou JC, Hirsch HH, Wali R, Crowder C, Nogueira J et (2004) alhistological patterns of polyomavirus nephropathy: Correlation , JGPT. All Rights Reserved 585

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