*3.3.1 Morphological characteristics of BKN*

BKN is morphologically characterized by intrarenal viral replication, mainly in tubular epithelial cell nuclei (intranuclear inclusions), causing tubular injury, shedding of tubular epithelial cells, and cell lysis (**Figures 7** and **8**). On immunofluorescence, focal immune complex-type granular deposition of IG along the tubular basement membrane is sometimes found, indicating BK infection (**Figure 9**), although the biologic and clinical significance of this finding needs further evaluation [5].

Viral replication in tubular epithelial cells can induce various nuclear changes: an amorphous ground-glass inclusion body (type 1), a central irregular inclusion body surrounded by a halo (type 2), finely granular nuclear alterations (type 3), and vesicular changes with coarsely clumped viral inclusions (type 4) (**Figure 10**).

#### **Figure 7.**

*BK virus nephropathy. Virally induced tubular epithelial cell injury and lysis in cortex (A, HE, 200x) and medulla (B, HE, 100x). Intranuclear viral inclusion bodies are observed (arrow).*

#### **Figure 8.**

*Virally induced tubular epithelial cells with intranuclear inclusions, shedding of infected cells, tubular injury, and lysis (A,HE, 400x and B, HE, 200x).*

In rare cases, the ascending PV infection can affect the parietal epithelial cells of Bowman's capsule, mainly detected by immunohistochemistry (**Figure 6**).

Diagnostic confirmation can easily be achieved by immunohistochemistry (**Figure 6**) or immunofluorescence, with antibodies directed against the polyomavirus T antigen, VP capsid proteins, or detection of intracellular virions of 40–50 nm in diameter by electron microscopy (**Figure 11**) [5, 57].

In early stages of PVN with focal and minimal tubular changes without tubular injury and characteristic intranuclear inclusions, a diagnosis can only be established by immunohistochemistry with antibody directed against SV-40-T antigen (**Figure 12**). Later in the course of the disease, many cases of PVN may show numerous infected cells and an inflammatory lymphocytic infiltrate with tubulitis mimicking acute T-cell-mediated rejection (**Figure 13**). Advanced disease, detected late after transplantation, often shows marked interstitial fibrosis/ tubular atrophy, while interstitial inflammation and viral replication may be variable (**Figure 14**).

#### **3.4 Differential diagnosis of BKN**

PVN must be differentiated from other rare viral infections, including CMV, herpes simplex virus, and adenovirus. CMV disease in transplant recipients is more frequent than BKN and usually affects the intestine, liver, or lungs but only rarely manifests as CMV reactivation in renal graft. Since the histological features of BKN

**53**

**Figure 10.**

**Figure 9.**

*membrane is sometimes found.*

infection are shown in **Table 2**.

may overlap with other viral infections, specific immunohistochemical staining is a sensitive tool for differentiating among BK, CMV, adenovirus, or herpes simplex viral infection. The main histological features of common transplant kidney viral

*Various nuclear changes induced by viral replication: type 1, an amorphous ground-glass inclusion body (A); type 2, a central irregular inclusion body surrounded by nuclear halo (B); type 3, finely granular nuclear alteration (C); type 4, vesicular nuclear changes with coarsely clumped viral inclusions (D, all HE, 600x).*

*On immunofluorescence, focal immune complex-type granular deposition of IgG along the tubular basement* 

*Viral Infections after Kidney Transplantation: CMV and BK*

*DOI: http://dx.doi.org/10.5772/intechopen.86043*

#### **Figure 9.**

*Perioperative Care for Organ Transplant Recipient*

In rare cases, the ascending PV infection can affect the parietal epithelial cells of Bowman's capsule, mainly detected by immunohistochemistry (**Figure 6**). Diagnostic confirmation can easily be achieved by immunohistochemistry (**Figure 6**) or immunofluorescence, with antibodies directed against the polyomavirus T antigen, VP capsid proteins, or detection of intracellular virions of

*Virally induced tubular epithelial cells with intranuclear inclusions, shedding of infected cells, tubular injury,* 

In early stages of PVN with focal and minimal tubular changes without tubular injury and characteristic intranuclear inclusions, a diagnosis can only be established by immunohistochemistry with antibody directed against SV-40-T antigen (**Figure 12**). Later in the course of the disease, many cases of PVN may show numerous infected cells and an inflammatory lymphocytic infiltrate with tubulitis mimicking acute T-cell-mediated rejection (**Figure 13**). Advanced disease, detected late after transplantation, often shows marked interstitial fibrosis/ tubular atrophy, while interstitial inflammation and viral replication may be

PVN must be differentiated from other rare viral infections, including CMV, herpes simplex virus, and adenovirus. CMV disease in transplant recipients is more frequent than BKN and usually affects the intestine, liver, or lungs but only rarely manifests as CMV reactivation in renal graft. Since the histological features of BKN

40–50 nm in diameter by electron microscopy (**Figure 11**) [5, 57].

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variable (**Figure 14**).

**Figure 8.**

**3.4 Differential diagnosis of BKN**

*and lysis (A,HE, 400x and B, HE, 200x).*

*On immunofluorescence, focal immune complex-type granular deposition of IgG along the tubular basement membrane is sometimes found.*

#### **Figure 10.**

*Various nuclear changes induced by viral replication: type 1, an amorphous ground-glass inclusion body (A); type 2, a central irregular inclusion body surrounded by nuclear halo (B); type 3, finely granular nuclear alteration (C); type 4, vesicular nuclear changes with coarsely clumped viral inclusions (D, all HE, 600x).*

may overlap with other viral infections, specific immunohistochemical staining is a sensitive tool for differentiating among BK, CMV, adenovirus, or herpes simplex viral infection. The main histological features of common transplant kidney viral infection are shown in **Table 2**.

#### **Figure 11.**

*Intranuclear viral inclusions in a tubular epithelial cell (A). Intranuclear virions measuring 40–50 nm in diameter (B, electron micrographs).*

#### **Figure 12.**

*BKN grade 1. Early phase with only focal tubular injury.(A, HE, 100x) and few SV-40 positive cells on immunohistochemistry (B, SV-40, 100x).*

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rejection.

**Figure 13.**

**3.5 Course of BKN**

*Viral Infections after Kidney Transplantation: CMV and BK*

However, the most important differential diagnosis, particularly in PVN after reduction of immunosuppression, remains T-cell-mediated acute rejection [6]. Careful correlations with clinical data, such as the presence of donor-specific antibodies, recent immunosuppression reduction, DNA viral load in the serum, and presence of decoy cells in the urine, provide additional information in order to make a correct diagnosis. Glomeruli and vessels must be carefully examined in order to exclude glomerulitis and vasculitis, which would strongly suggest concomitant rejection. C4d positivity and diffuse peritubular capillaritis outside the area of extensive interstitial inflammation, together with positive donorspecific antibodies (DSA), are consistent with concomitant antibody-mediated

*indistinguishable from acute rejection. There was no endarteritis (A, PAS, 200x). Numerous BK-positive cells* 

*BKN grade 2. Florid phase with severe interstitial inflammation and tubulitis in BK nephropathy,* 

*on immunohistochemistry were detected. (SV-40, 200×).*

A diagnosis of PVN and concomitant T-cell-mediated rejection after immunosuppression reduction is challenging and needs careful correlation of biopsy findings with the dynamics of BK viremia. Focal interstitial inflammation in the context of stable graft function and recently cleared BK viremia should be interpreted as residual BKN, but the same histology findings detected beyond 3 months after BK clearance, accompanied by a rise in serum creatinine, might rather point toward acute rejection.

The natural course of BKN remains to be elucidated. Some authors have reported that biopsies obtained after reduction of immunosuppression during

*DOI: http://dx.doi.org/10.5772/intechopen.86043*

*Viral Infections after Kidney Transplantation: CMV and BK DOI: http://dx.doi.org/10.5772/intechopen.86043*

#### **Figure 13.**

*Perioperative Care for Organ Transplant Recipient*

*Intranuclear viral inclusions in a tubular epithelial cell (A). Intranuclear virions measuring 40–50 nm in* 

*BKN grade 1. Early phase with only focal tubular injury.(A, HE, 100x) and few SV-40 positive cells on* 

**54**

**Figure 12.**

*immunohistochemistry (B, SV-40, 100x).*

**Figure 11.**

*diameter (B, electron micrographs).*

*BKN grade 2. Florid phase with severe interstitial inflammation and tubulitis in BK nephropathy, indistinguishable from acute rejection. There was no endarteritis (A, PAS, 200x). Numerous BK-positive cells on immunohistochemistry were detected. (SV-40, 200×).*

However, the most important differential diagnosis, particularly in PVN after reduction of immunosuppression, remains T-cell-mediated acute rejection [6]. Careful correlations with clinical data, such as the presence of donor-specific antibodies, recent immunosuppression reduction, DNA viral load in the serum, and presence of decoy cells in the urine, provide additional information in order to make a correct diagnosis. Glomeruli and vessels must be carefully examined in order to exclude glomerulitis and vasculitis, which would strongly suggest concomitant rejection. C4d positivity and diffuse peritubular capillaritis outside the area of extensive interstitial inflammation, together with positive donorspecific antibodies (DSA), are consistent with concomitant antibody-mediated rejection.

A diagnosis of PVN and concomitant T-cell-mediated rejection after immunosuppression reduction is challenging and needs careful correlation of biopsy findings with the dynamics of BK viremia. Focal interstitial inflammation in the context of stable graft function and recently cleared BK viremia should be interpreted as residual BKN, but the same histology findings detected beyond 3 months after BK clearance, accompanied by a rise in serum creatinine, might rather point toward acute rejection.

#### **3.5 Course of BKN**

The natural course of BKN remains to be elucidated. Some authors have reported that biopsies obtained after reduction of immunosuppression during

#### **Figure 14.**

*BKN grade 3. Moderate interstitial fibrosis/tubular atrophy and interstitial inflammation composed of CD3 positive lymphocytes in areas of fibrosis (A, CD3 and PAS, 100x). Many tubules show viral replication (B, SV-40 antigen, 200x).*

decrease of the plasma viral load may show severe interstitial infiltrate and tubulitis reminiscent of T-cell-mediated acute rejection, but the outcome of renal grafts was good despite prolonged reduction of immunosuppression without corticosteroid administration [4, 6, 58, 59]. Such patients typically presented with a transient increase in serum creatinine, accompanied by a decrease in plasma viral load, which finally disappeared [59]. Moreover, serum creatinine returned to the baseline level after a few months. In subsequent biopsies, the virus was cleared from renal tissue, and inflammation resolved without the presence of marked interstitial fibrosis. These authors have suggested that such tubulointerstitial nephritis might be immune reconstitution-associated graft inflammation, enabling the resolution of PVN.

**57**

investigation [6].

Viral replication tubules

Interstitial inflammation

**Table 2.**

*Viral Infections after Kidney Transplantation: CMV and BK*

Viral inclusions Type 1: an amorphous ground-

alteration

alteration

Endothelial cells No Yes Inflammatory cells No Yes Acute tubular injury Rarely Rarely

Tubulitis Mild to severe Mild

*Histologic features of CMV and polyoma BK viral lesions in transplanted kidney.*

glass inclusion body

Type 2: a central irregular inclusion body surrounded by nuclear halo Type 3: finely granular nuclear

Type 4: finely granular nuclear

Yes Yes

Focal to diffuse Focal

The challenging concepts of immune reconstitution injury and extensive inflammation in resolving BKN after reducing immunosuppression need further

**Polyomavirus Cytomegalovirus**

Smudgy/ground-glass nuclear inclusions surrounded by typical halo-owl eye

Various studies have indicated that different extents of BKN in the transplant may predict the clinical presentation and outcome of the disease [58, 60, 61].

responsible for irreversible tissue injury leading to graft failure [5, 47, 62].

with worsening of kidney function and graft failure in 50% of cases (**Table 3**).

transiently, particularly when only the medulla is involved [5].

impact on the prognosis of the disease and therefore on allograft survival. Early diagnosis of PVN is difficult, because early BKN stage does not show any signs of systemic infection, proteinuria, or hematuria. Renal function may remain normal

In order to provide optimal diagnostic and prognostic information of BKN, the Banff working group on BKN proposed three clinically significant disease grades based on the severity of polyomavirus replication and the degree of interstitial fibrosis [47, 62, 63]. BK virus replication was defined as the histologic viral load, estimated by the % of virally infected epithelial cells detected by immunohistochemistry. It ranged from scattered SV-40-positive cells in BKN grade 1 to numerous in grades 2 and 3 (**Figures 12–14**). In addition to SV-40-positive cells, grade 3 is characterized by interstitial fibrosis, which is

Disease grade may reflect the time of the diagnosis: BKN grade 1 was generally diagnosed in the first 5 months after transplantation, usually presenting with normal renal function and associated with a favorable outcome in 85–90% of cases. In contrast, grade 2 BKN was detected 6–12 months posttransplantation, characterized by elevated serum creatinine or acute graft injury leading to graft failure in 25% of cases. Finally, BKN grade 3 was usually detected more than 12 months after transplantation, also associated

Since BKN has limited treatment options, the early detection of PVN has a major

**3.6 Clinical presentation and management of BKN**

*3.6.1 Clinical presentation and prognosis*

*DOI: http://dx.doi.org/10.5772/intechopen.86043*


#### **Table 2.**

*Perioperative Care for Organ Transplant Recipient*

decrease of the plasma viral load may show severe interstitial infiltrate and tubulitis reminiscent of T-cell-mediated acute rejection, but the outcome of renal grafts was good despite prolonged reduction of immunosuppression without corticosteroid administration [4, 6, 58, 59]. Such patients typically presented with a transient increase in serum creatinine, accompanied by a decrease in plasma viral load, which finally disappeared [59]. Moreover, serum creatinine returned to the baseline level after a few months. In subsequent biopsies, the virus was cleared from renal tissue, and inflammation resolved without the presence of marked interstitial fibrosis. These authors have suggested that such tubulointerstitial nephritis might be immune reconstitution-associated graft inflammation,

*BKN grade 3. Moderate interstitial fibrosis/tubular atrophy and interstitial inflammation composed of CD3 positive lymphocytes in areas of fibrosis (A, CD3 and PAS, 100x). Many tubules show viral replication* 

**56**

**Figure 14.**

*(B, SV-40 antigen, 200x).*

enabling the resolution of PVN.

*Histologic features of CMV and polyoma BK viral lesions in transplanted kidney.*

The challenging concepts of immune reconstitution injury and extensive inflammation in resolving BKN after reducing immunosuppression need further investigation [6].

## **3.6 Clinical presentation and management of BKN**

#### *3.6.1 Clinical presentation and prognosis*

Various studies have indicated that different extents of BKN in the transplant may predict the clinical presentation and outcome of the disease [58, 60, 61].

In order to provide optimal diagnostic and prognostic information of BKN, the Banff working group on BKN proposed three clinically significant disease grades based on the severity of polyomavirus replication and the degree of interstitial fibrosis [47, 62, 63]. BK virus replication was defined as the histologic viral load, estimated by the % of virally infected epithelial cells detected by immunohistochemistry. It ranged from scattered SV-40-positive cells in BKN grade 1 to numerous in grades 2 and 3 (**Figures 12–14**). In addition to SV-40-positive cells, grade 3 is characterized by interstitial fibrosis, which is responsible for irreversible tissue injury leading to graft failure [5, 47, 62].

Disease grade may reflect the time of the diagnosis: BKN grade 1 was generally diagnosed in the first 5 months after transplantation, usually presenting with normal renal function and associated with a favorable outcome in 85–90% of cases. In contrast, grade 2 BKN was detected 6–12 months posttransplantation, characterized by elevated serum creatinine or acute graft injury leading to graft failure in 25% of cases. Finally, BKN grade 3 was usually detected more than 12 months after transplantation, also associated with worsening of kidney function and graft failure in 50% of cases (**Table 3**).

Since BKN has limited treatment options, the early detection of PVN has a major impact on the prognosis of the disease and therefore on allograft survival. Early diagnosis of PVN is difficult, because early BKN stage does not show any signs of systemic infection, proteinuria, or hematuria. Renal function may remain normal transiently, particularly when only the medulla is involved [5].


#### **Table 3.**

*Characteristics of different BKN grades regarding viral load, chronic tissue injury-interstitial fibrosis, renal function, time of diagnosis after transplantation, and outcome.*

#### *3.6.2 Screening of PVN*

To date, reduction of baseline immunosuppression remains the only potentially effective therapeutic strategy of BKN, but it is associated with an increased risk of rejection. It is considered that preemptive reduction of immunosuppression prior to the development of overt nephropathy might be beneficial [6, 51, 59]. Since unrecognized BKN diagnosed late after transplantation causes chronic tissue injury and graft failure, the goal of screening protocols and classification schemes of BKN is to characterize early disease grades that respond to therapeutic intervention and may heal without progressing to chronic graft injury.

The first step of viral reactivation shown in almost all patients is characterized by the detection of characteristic polyomavirus inclusion-bearing cells in the urine—decoy cells (**Figure 15**). Initial viruria may be followed by detection of BK virus in plasma and onset of BKN after a 6–12-week window in some patients but only in a minority (**Figure 16**) [51].

Current guidelines recommend a urinary cytology test in order to detect urinary decoy cells initially and then a plasma test by PCR if urinary decoy cells are consistently present [51]. While PVN is most commonly diagnosed in the first year after transplantation, urine screening at least every 3 months during the first 2 years and after antirejection treatment seems appropriate to cover the majority of PVN cases [51]. The cytology urine test is characterized by a high negative predictive value to rule out a diagnosis of BKN and reduce costs. In addition, a window between viral reactivation and BKN enables urine samples to be screened in time.

However, several studies have shown that only a variable number of patients with urinary shedding of virus progressed to BKN. Notably, BK viruria and even

**59**

*Viral Infections after Kidney Transplantation: CMV and BK*

viremia may represent transient asymptomatic BK activation or may originate from extrarenal sites, usually along the lower urinary tract. In patients without biopsyproven BKN, preemptive long-lasting reduction of immunosuppression could be

A plasma test by PCR detecting BK copies is currently the accepted biomarker for clinical application, although the exact range of viral load that would predict BKN cannot be defined. The majority of patients with more than 10,000 copies per ml DNA in 1 ml of plasma show BKN on renal biopsy, but some patients with hardly detectable BK virus copy numbers may have manifest BKN. Several studies have indicated that PCR-based BK viremia correlates only moderately well with the presence of BKN and severity of the intrarenal disease, ranging between 25 and

Several biomarkers had been proposed in order to enable noninvasive diagnosis of definitive BKN without the risk of renal biopsy; these include heat shock protein 90alfa, CXCL9, neutrophil gelatinase-associated lipocalin, urinary exosomal

Polyomavirus-Haufen are tight cast-like three-dimensional viral aggregates, detected by negative staining electron microscopy of a voided urine sample. Since polyomavirus-Haufen admixed with uromodulin is formed in the tubular lumens, they might specifically predict intrarenal disease, comparable to renal biopsy [68]. Recent studies have indicated that the titer of polyomavirus-Haufen tightly correlates with the degree of intrarenal polyomavirus replication, providing additional information on the severity of PVN [64]. The urinary polyomavirus-Haufen test may emerge as a sensitive and specific biomarker for intrarenal viral disease, with positive and negative predictive value higher than 90%. The limitations of this

potentially harmful due to increased risk of acute rejection [64].

*Type and prevalence of BK virus (BKV) infections in kidney transplant recipients.*

biomarkers, urinary VP1, and urinary Haufen [65–67].

*3.6.3 Biomarkers of BKN*

**Figure 16.**

75% (**Figure 15**) [6, 57].

*DOI: http://dx.doi.org/10.5772/intechopen.86043*

**Figure 15.** *Decoy cells in urine screening test.*

*Perioperative Care for Organ Transplant Recipient*

SV-40 positive cells

Grade 2 Numerous Less than

Grade 3 Numerous More than

**Viral load Interstitial** 

**fibrosis**

25%

25%

*3.6.2 Screening of PVN*

**Table 3.**

**PVN disease grade**

Grade 1 Scattered

heal without progressing to chronic graft injury.

*function, time of diagnosis after transplantation, and outcome.*

only in a minority (**Figure 16**) [51].

To date, reduction of baseline immunosuppression remains the only potentially effective therapeutic strategy of BKN, but it is associated with an increased risk of rejection. It is considered that preemptive reduction of immunosuppression prior to the development of overt nephropathy might be beneficial [6, 51, 59]. Since unrecognized BKN diagnosed late after transplantation causes chronic tissue injury and graft failure, the goal of screening protocols and classification schemes of BKN is to characterize early disease grades that respond to therapeutic intervention and may

*Characteristics of different BKN grades regarding viral load, chronic tissue injury-interstitial fibrosis, renal* 

Increased serum creatinine, renal failure

Increased serum creatinine, acute renal failure

**Renal function Time of diagnosis** 

No Normal 4–5 85–90

**after TX (months)**

6–12 75

12 50

**Favorable outcome (%)**

The first step of viral reactivation shown in almost all patients is characterized by the detection of characteristic polyomavirus inclusion-bearing cells in the urine—decoy cells (**Figure 15**). Initial viruria may be followed by detection of BK virus in plasma and onset of BKN after a 6–12-week window in some patients but

reactivation and BKN enables urine samples to be screened in time.

Current guidelines recommend a urinary cytology test in order to detect urinary decoy cells initially and then a plasma test by PCR if urinary decoy cells are consistently present [51]. While PVN is most commonly diagnosed in the first year after transplantation, urine screening at least every 3 months during the first 2 years and after antirejection treatment seems appropriate to cover the majority of PVN cases [51]. The cytology urine test is characterized by a high negative predictive value to rule out a diagnosis of BKN and reduce costs. In addition, a window between viral

However, several studies have shown that only a variable number of patients with urinary shedding of virus progressed to BKN. Notably, BK viruria and even

**58**

**Figure 15.**

*Decoy cells in urine screening test.*

**Figure 16.** *Type and prevalence of BK virus (BKV) infections in kidney transplant recipients.*

viremia may represent transient asymptomatic BK activation or may originate from extrarenal sites, usually along the lower urinary tract. In patients without biopsyproven BKN, preemptive long-lasting reduction of immunosuppression could be potentially harmful due to increased risk of acute rejection [64].
