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NDT Advance Access published online on May 25, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn289
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Monitoring of BK virus replication in the first year following renal transplantation

Cristina Costa1,*, Massimiliano Bergallo1,*, Sara Astegiano1, Maria Elena Terlizzi1, Francesca Sidoti1, Giuseppe P. Segoloni2 and Rossana Cavallo1

1 Dipartimento di Sanità Pubblica e Microbiologia, Laboratorio di Virologia, Università di Torino 2 Dipartimento di Medicina Interna, Unità Trapianto Rene, Ospedale Molinette, Torino, Italy

Correspondence and offprint requests to: Cristina Costa, Dipartimento di Sanità Pubblica e Microbiologia, Laboratorio di Virologia, Università di Torino, Italy. E-mail: cristina.costa{at}unito.it, rossana.cavallo{at}unito.it



   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. BK virus-associated nephropathy (BKVAN) is one of the most common viral diseases affecting renal allografts. Screening for viral replication may allow for earlier intervention with reduced allograft loss. A plasma viral load >104 copies/mL of BKV DNA is recommended for a presumed diagnosis of BKVAN.

Methods. We monitored BKV load on serum and urine samples by Real-Time TaqMan PCR in 229 renal transplant recipients in the first year post-transplantation. Overall, 2025 serum and 2025 urine samples were evaluated. A graft biopsy was performed in 47/229 patients to investigate the declining renal function. Operating characteristics [sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV)] and receiver operating characteristic (ROC) curve analysis at different viral load values were calculated.

Results. Serum BKV viral load was >104 in 5/229 patients (2.2%). A histological diagnosis of BKVAN was made in 3/229 patients (1.3%): 3/5 (60.0%) among those with serum viral load >104 and 3/4 (75.0%) in those with >1.6 x 104. Operating characteristics of a serum BK load of 104 for the diagnosis of BKVAN were as follows: sensitivity, 100%; specificity, 99.1%; NPV, 100%; PPV, 59.4%. Specificity and PPV rose to 99.6% and 75.0% when using a cut-off level of 1.6 x 104 copies/mL.

Conclusions. The recommended level of BK viraemia of 104 copies/mL is useful to identify patients at risk of BKVAN, although specificity and PPV increase by using a cut-off level of 1.6 x 104 copies/mL. BK replication may occur in the first 3 months post-transplantation and subsequently recede. Therefore, the temporal profile of BKV replication has to be accurately evaluated and occasionally elevated values should prompt a closer monitoring.

Keywords: BKV-associated nephropathy; monitoring; polyomavirus BK; renal transplantation



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Human polyomavirus BK (BKV) is a worldwide-distributed virus the seroprevalence rate of which is ~70–90% in adults. Following primary infection, BK remains latent in the renourinary tract as the epidemiologically most relevant latency site, and in B cell, brain, spleen and probably other tissues. Reactivation with asymptomatic viruria may occur in both immunocompetent subjects and immunocompromised patients. In renal transplantation, in the context of intense immunosuppression, viral replication may determine BKV-associated nephropathy (BKVAN) with interstitial nephritis and/or ureteral stenosis in 1–10% of the patients, particularly in the first year following transplantation, and leading to graft failure and return to haemodialysis in 30 to 80% of the cases [1–3]. Today, BKVAN is one of the most common viral diseases affecting renal allografts. Screening for polyomavirus replication may allow for earlier intervention with reduced allograft loss [2]. A presumed diagnosis of BKVAN may be made in the presence of surrogate markers of viral replication, such as plasma viral load >104 copies/mL of BKV DNA [2]. Thus, screening for BKV replication represents the basic strategy to predict early the onset of BKVAN [2,3]. The aim of this study was to investigate the operating characteristics of BKV replication monitoring for the development of BKVAN in renal transplant recipients in the first year post-transplantation.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients and samples
Two hundred twenty-nine renal transplant recipients (140 males, 89 females; mean age ± standard deviation, 53.2 ± 14.3 years) were studied in the first year post-transplantation. BKV replication monitoring was performed on serum and urine samples twice monthly in the first 3 months, and then every 3 months; in the presence of renal function abnormalities or on the basis of clinical judgement, intervals of monitoring were reduced to twice monthly. A complete monitoring was available for 174 patients. Overall, 2025 serum and 2025 urine samples were studied at the Virology Unit of the University of Turin, Italy. The findings of two or more consecutive positive serum or urine samples were defined as sustained viraemia and viruria. Immunosuppressive protocols were as follows: for induction basiliximab (B), mycophenolate mofetil (MMF) and steroid (S) in 75 patients; B, MMF and tacrolimus (FK506) in 10; B, MMF, S and FK506 in 63; B, FK506 and S in 72; B, MMF, S and cyclosporine A (CyA) in 1; B, S and antilymphocyte globulin (ALG) in 3; B, S, MMF and sirolimus in 2; B, S, MMF and CyA in 3; and for maintenance: FK506 and S in 92 patients; FK506, MMF and S in 118; FK506 and sirolimus in 1; CyA and S in 5; S, CyA and MMF in 13. In the presence of BK viraemia, a reduction of immunosuppression level was made.

Clinical charts of all the patients and the database of the Regional Reference Transplant Centre were reviewed. During the study period, 47 of 229 patients underwent at least one kidney allograft biopsy (overall 63 procedures) to investigate the declining renal function. The occurrence of BKVAN was diagnosed by histopathological evaluation on formalin-fixed and paraffin-embedded 4-µ sections, using periodic acid-Schiff, Masson's trichrome, phosphotungstic acid haemotoxylin and acid fucsin-orange G stains. Moreover, immunohistochemistry with immunoperoxidase staining was performed on fixed material using polyclonal anti-SV40 antibody (dilution 1:20 000; Lee Biomolecular Research Labs, San Diego CA, USA).

Thirty healthy subjects, including health care workers in the Nephrology Ward (8 subjects) or in the Virology Unit (4 subjects), and 50 non-transplant patients with lupus nephritis (14 not treated and 36 treated with steroids or immunosuppressive agents, as described elsewhere [4]) were also evaluated.

PCR for BKV-DNA quantification in serum and urine samples
Extraction procedure from serum and urine samples was performed as previously described [5]. BKV-DNA quantification was performed by Real-Time TaqMan PCR using a commercial kit (BKV Q-PCR Alert Kit; Nanogen Advanced Diagnostic, Milano, Italy) for the detection of the target viral gene encoding for the large T-antigen of BKV with the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). According to the manufacturer's instructions, PCR amplifications were set up in a reaction volume of 25 µL that contained 5 µL of extracted sample or negative control (sterile double-distilled H2O) or plasmid. Thermal cycling was as follows: 50°C for 2 min, an initial denaturation step at 95°C for 10 min that was followed by 45 cycles at 95°C for 15 s (denaturation) and 60°C for 1 min (reannealing and extension). Standard curves for the quantification of DNA were constructed by plotting the threshold cycle against the logarithm of serial 10-fold dilutions, ranging from 102 to 105, of the corresponding plasmid. Amplification data were analysed by the Sequence Detection System software (Applied Biosystems). Each sample was subjected to simultaneous TaqMan PCR for the housekeeping gene human β-globin; results were considered acceptable only in the presence of β-globin positivity. The assay was linear in the range 102–105 copies per reaction, corresponding to an absolute quantification of 103 copies/mL.

Different values for serum viral load (ranging from 3 x 103 to 1.6 x 104 copies/mL) were investigated as markers for possible evolution towards BKVAN or presumed BKVAN, as appropriate.

Statistical analysis
Operating characteristics, including sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and receiver operating characteristic (ROC) curve analysis, were evaluated by using commercially available software (MedCalc; version 9.2.1.0).



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Results are summarized in Table 1. Real-time PCR for BKV DNA was positive (i.e. >103 copies/mL) in 93 of 2025 (4.6%) serum samples, obtained from 21 of 229 patients (9.2%), and in 228 of 2025 (11.3%) urine samples, obtained from 42 of 229 patients (18.3%). Sustained viraemia and viruria were found in 11 and 19 patients, respectively, with a mean time to becoming negative of 34.7 ± 23.0 days (mean ± SD). Considering serum samples, BKV viral load was >104 in 36/2025 specimens (1.8%), from 5/229 patients (2.2%). A histologically confirmed diagnosis of BKVAN was made in three of these patients, giving an overall BKVAN prevalence of 1.3% in the 229 kidney transplant recipients and 60% (3/5) among patients with serum viral load >104. Graft loss subsequently occurred in one of these three patients (33.3%). Considering the 42 remaining patients in which a renal biopsy was performed, 2 presented a serum viral load <3 x 103 and the others were negative. None of them presented histopathological findings associated with BKVAN. Two patients presented a serum viral load >104 in the first month following transplantation; however, with reduction of immunosuppression, the viral loads subsequently reduced at the following determinations 1 week later until they returned negative or <3 x 103 three weeks later. Operating characteristics of a serum BK load of 104 for the diagnosis of BKVAN were as follows: sensitivity, 100%; specificity, 99.1%; NPV, 100%; and PPV, 59.4%. BK serum viral load was >1.6 x 104, >1.3 x 104, >7 x 103, >5 x 103 and >3 x 103 in 4/229 (1.7%), 5/229 (2.2%), 6/229 (2.6%), 11/229 (4.8%) and 14/229 (6.1%) patients, respectively. Among patients with viraemia >1.6 x 104, three of four (75%) developed BKVAN. The area under the ROC curve was 0.997 (95% confidence interval, 0.978–0.999) (Figure 1). Considering urine samples, a BK viral load >107 copies/mL in relation to a diagnosis of BKVAN, the following operating characteristics were evidenced: sensitivity, 100%; specificity, 95.5%, NPV, 100% and PPV, 27.3%. Operating characteristics for all the cut-off values are reported in Table 1. Regarding the temporal profile, usually viruria preceded BK viraemia by 1.5 months (mean ± standard deviation, 42.1 ± 30.1 days), although levels <105 copies/mL could also be detected in the absence of subsequent detection of viraemia, while viraemia never occurred in the absence of viruria.


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  		Table 1 Operating characteristics of BKV-DNA quantification in serum and urine samples in relation to a histologically confirmed diagnosis of BKVAN in 229 renal transplant patients

 

Figure 1
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  		Fig. 1 ROC curve analysis to predict BK virus-associated nephropathy for serum BK viral load in 229 renal transplant patients. Area under the ROC curve, 0.997 (95% confidence interval, 0.978–0.999).

 
Considering healthy controls, BKV DNA was positive in 4/30 (13.3%; viral load >104 copies/mL but <1.3 x 104 copies/mL in all of them) and in 5/30 (16.7%; viral load <107 in all of them) of serum and urine samples, respectively; considering non-transplant patients with lupus nephritis, BKV DNA was positive in 3/50 (6.0%; viral load >104 copies/mL but <1.3 x 104 copies/mL in one) and in 20/50 (40.0%; viral load <107 in all of them) in serum and urine samples, respectively.



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The aim of this study was to investigate the operating characteristics of BKV replication monitoring for the development of BKVAN in renal transplant recipients in the first year post-transplantation.

By 1 year post-transplantation, 21 patients (9.2%) developed viraemia and 42 (18.3%) viruria. While cumulative incidence of BK viraemia was similar to that reported in other studies, ranging from 9 to 40% [6,7,3,8], viruria occurred at a lower frequency in comparison to that of other studies, ranging from 24 to 40% [7,3,8]. This could be attributable to different follow-up sampling timings (e.g. twice monthly in the first 3 months in our study versus weekly in others [7]), thus missing episodes of transient viruria, micturition intervals and fluctuations of urine content [2], although it is to note that the difference between our data on viruria and those of others [8] is not statistically significant. Considering the prevalence of BK viruria and viraemia in healthy controls, our results seem to be in contrast with those obtained from other studies [10]. We could speculate that these data are related to a major susceptibility to BKV contact in our control group, including mainly Nephrology and Virology operators, as previously demonstrated for Blood Transfusion Centre operators [11]; while considering the occurrence of BK viraemia and viruria in non-transplant patients with lupus nephritis, this has been already discussed elsewhere [4].

The identification of a relevant threshold of BK viraemia or viruria for a presumed diagnosis of BKVAN represents an important issue in clinical management of renal transplant recipients [12]. A multidisciplinary international panel of experts recommends screening for BKV replication on urine every 3 months in the first 2 years post-transplantation and yearly thereafter [2]. Viruria can be studied either through urine cytology (decoy cells) or quantification of viral load. Although decoy cell evaluation has a high NPV, as their absence virtually excludes BKVAN, it is to note that sensitivity and PPV are low and cannot differentiate between human polyomavirus BK and JC [13]. Regarding urine BK load, there is general agreement that repeated values >107 copies/mL are associated with BKVAN, while the detection of BK viraemia >104 copies/mL is considered the most reliable surrogate marker of presumed BKVAN in renal transplant recipients. The results obtained in this study concur with the internationally recommended cut-off levels. It is to note that the adoption of a cut-off level of 1.6 x 104, as recently suggested in a study by Viscount et al. [8], ameliorates operating characteristics, particularly in terms of PPV (Table 1). This has also to be interpreted considering the time from transplantation, as our study focused on the first year post-transplantation, while in the study by Viscount et al., samples were collected at a median of 2 years after transplantation. Moreover, like most studies investigating BKVAN, our study was limited by the low number of affected patients.

A definitive diagnosis of BKVAN is made by the demonstration of typical viral cytopathic findings at histopathology. However, given the focal involvement of early BKVAN and the possibility of tissue-sampling errors, clinical management is often based on the surrogate markers of viral replication. On the other hand, the requirement of tissue evaluation in patients who are suspected to have BKVAN remains, also taking into account that a renal biopsy is necessary to exclude other pathologic processes, such as acute rejection that may coexist [14].

Quantification of viraemia and viruria is mandatory in the follow-up of kidney graft recipients, as evidenced by the analysis of operating characteristics on a large group of patients [2]. It has to be taken into consideration that BK replication may occur in the first 3 months following transplantation and may subsequently recede, probably due to the early development of a specific cellular immune response [15,16], as in immunocompetent subjects. Therefore, the temporal profile of BKV replication has to be accurately evaluated and occasionally elevated values should prompt a closer monitoring in order to be confirmed, as well as the modulation of immunosuppressive therapy.

Conflict of interest statement. None declared.



   Notes
 
* Cristina Costa and Massimiliano Bergallo equally contributed to this work and share first authorship. Back



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Nickeleit V, Singh HK, Mihatsch MJ. Polyomavirus nephropathy: morphology, pathophysiology, and clinical management. Curr Opin Nephrol Hypertens (2003) 12:599–605.[Web of Science][Medline]
  2. Hirsch HH, Brennan DC, Drachenberg CB, et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation (2005) 79:1277–1286.[CrossRef][Web of Science][Medline]
  3. Hariharan S. BK virus nephritis after renal transplantation. Kidney Int (2006) 69:655–662.[CrossRef][Web of Science][Medline]
  4. Colla L, Mesiano P, Morellini V, et al. Human polyomavirus BK in patients with lupus nephritis: clinical and histological correlations. Lupus (2007) 16:881–886.[Abstract/Free Full Text]
  5. Bergallo M, Costa C, Gribaudo G, et al. Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples. New Microbiol (2006) 29:111–119.[Medline]
  6. Hirsch HH, Knowles W, Dickenmann M, et al. Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients. N Engl J Med (2002) 347:488–496.[Abstract/Free Full Text]
  7. Brennan DC, Agha I, Bohl DL, et al. Incidence of BK with tacrolimus versus cyclosporine and impact of preemptive immunosuppression reduction. Am J Transplant (2005) 5:582–594.[CrossRef][Web of Science][Medline]
  8. Viscount HB, Eid AJ, Espy MJ, et al. Polyomavirus polymerase chain reaction as a surrogate marker of polyomavirus-associated nephropathy. Transplantation (2007) 84:340–345.[CrossRef][Web of Science][Medline]
  9. Sundsfjord A, Osei A, Rosenqvist H, et al. BK and JC viruses in patients with systemic lupus erythematosus: prevalent and persistent BK viruria, sequence stability of the viral regulatory regions, and nondetectable viremia. J Infect Dis (1999) 180:1–9.[CrossRef][Web of Science][Medline]
  10. Randhawa P, Uhrmacher J, Pasculle W, et al. A comparative study of BK and JC virus infections in organ transplant recipients. J Med Virol (2005) 238–243.
  11. Dolei A, Pietropaolo V, Gomes E, et al. Polyomavirus persistence in lymphocytes: prevalence in lymphocytes from blood donors and healthy personnel of a blood transfusion centre. J Gen Virol (2000) 81:1967–1973.[Abstract/Free Full Text]
  12. Pang XL, Doucette K, LeBlanc B, et al. Monitoring of polyomavirus BK viruria and viremia in renal allograft recipients by use of a quantitative real-time PCR assay: one-year prospective study. J Clin Microbiol (2007) 45:3568–3573.[Abstract/Free Full Text]
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  15. Binggeli S, Egli A, Schaub S, et al. Polyomavirus BK-specific cellular immune response to VP1 and large T-antigen in kidney transplant recipients. Am J Transplant (2007) 7:1131–1139.[CrossRef][Web of Science][Medline]
  16. Prosser SE, Orentas RJ, Jurgens L, et al. Recovery of BK virus large T-antigen-specific cellular immune response correlates with resolution of BK virus nephritis. Transplantation (2008) 85:185–192.[Web of Science][Medline]
Received for publication: 7. 2.08
Accepted in revised form: 24. 4.08


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This Article
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