Cytologic and biomolecular diagnosis of polyomavirus infection in urine specimens of HIV-positive patients

OBJECTIVE: To evaluate the frequency of human polyomavirus reactivation in urine specimens from HIV-positive patients; compare the sensitivity of cytology, immunohistochemistry and molecular biology; differentiate viral genotypes; and correlate the results with urinary cytologic abnormalities. STUDY DESIGN: Urine specimens from 78 unselected HIV-positive patients were evaluated by means of cytology, immunohistochemistry and nested polymerase chain reaction (n-PCR) to evaluate the presence of polyomaviruses. Restriction fragment length polymorphism (RFLP) was carried out in positive cases in order to differentiate BK virus (BKV) from JC virus (JCV). CD4 cells and serum creatinine levels were evaluated as indices of immune status and renal function, respectively, whereas the presence of red blood cells was used as an index of urogenital damage. RESULTS: Cytologic evidence of polyomavirus infection was found in 17 samples and immunohistochemically confirmed in 9; another 6 cytologically negative cases were detected by means of immunohistochemistry. In all cases, only one or two cells showed typical viral inclusions or positive staining. n-PCR identified 44 positive samples, thus confirming all of the cytologically and immunohistochemically positive cases and detecting polyomavirus genome in a further 21. RFLP detected 39 JCV, 1 BKV and 4 JCV-BKV infections. No correlation was found between the presence or type of polyomavirus and immune status, but red blood cells were found more frequently in the positive than in the negative samples. Serum creatinine levels fell within the normal range in all cases. CONCLUSION: Molecular biology is the most sensitive tool for detecting polyomavirus urinary infection in HIV-positive patients and the only reliable method of differentiating JCV and BKV viral genotypes.     

Real-time PCR test for detection and quantification of human polyomavirus BK (BKV) DNA

The human polyomavirus BK (BKV) is wide-spread pathogen, associated with urogenital tract disorders or even nephropathy in immunosuppressed patients. Nowadays molecular detection by real-time PCR (qPCR) is recognized as a method-of-choice for detecting human polyomaviruses in clinical samples. The aim of the study was development of real-time PCR assay for detection and quantification of polyomavirus BK DNA in clinical samples, using specific primers targeting a viral DNA VP3 gene and a TaqMan hydrolyzing probe. The analytical sensitivity of assay was tested using serial dilutions of BKV DNA in range between 13500 and 15 copies/ml. 27 urine samples and 23 plasma samples taken from a group of 22 adult recipients of allogeneic HSCT were tested for the presence of polyomavirus BK in the LightCycler system. Described in-house real-time PCR assay detected BKV DNA in 8 specimens (6 urine and 2 plasma). Detected average viral load was 170 copies/ml for plasma and 1250 copies/ml for urine samples, respectively. The results of this study show that developed TaqMan-based probe qPCR assay is very reliable and valuable for detection and quantification of BKV DNA, both in urine and plasma samples. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid diagnostics of polyomavirus BK infections in the clinical laboratory.     

Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipients

Objective:  BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients.
Patients and methods:  We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood.
Results:  Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR.
Conclusions:  Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation.     

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.     

Periodic assessment of urine and serum by cytology and molecular biology as a diagnostic tool for BK virus nephropathy in renal transplant patients

OBJECTIVE: To investigate the significance of polyomavirus (PV) viruria and viremia by morphologic, immunohistochemical and molecular analysis (multiplex nested-polymerase chain reaction) in renal transplant patients. STUDY DESIGN: Urine (n=328), serum (n= 53) and renal biopsies (n=24) from renal transplant patients (n=106) were studied. RESULTS: Decoy cells were found in 53 samples (16%) from 19 patients (18%); viral DNA was amplified in all urinary samples and disclosed BK virus (BKV) (n=24), JC virus (JCV) (n=16), and JCV and BKV DNA (n=13). BKV was the prevailing genotype in patients with a high frequency of decoy cell excretion (p = 0.001). JCV excretion correlated with a low number (p = 0.01) and BKV with a high number of decoy cells (p=0.003). PV DNA was amplified from 30/53 serum samples (56.6%); BKV was the prevailing genotype (p = 0.04). On 24 renal biopsies (18 from the decoy cell-negative and 6 from the decoy cell-positive group) PV nephropathy (PVN) was identified and BKV DNA amplified in 4 biopsies, all from the group with a high frequency of decoy cell excretion. PVN was not identified in renal biopsies from the decoy cell-negative group. CONCLUSION: PV infection is frequent in renal transplant patients. The BKV genotype in urine and serum is significantly related to a high frequency and high number of decoy cells. PVN occurs only in patients with BKV viremia and a high number and frequency of decoy cell excretion in urine. In the absence of decoy cells, PVN can be excluded. Cytologic analysis of urine is an important diagnostic tool for screening renal transplant patients at risk of PVN.     

Occurrence of the European subgroup of subtype I BK polyomavirus in Japanese-Americans suggests transmission outside the family

To examine the mode of transmission of BK polyomavirus (BKV), urine samples were collected from Japanese-Americans in Los Angeles and from other southern Californians. Subtype I was the main subtype found in samples from both groups. The subtype I subgroup Ib-2, which is predominant in Europe, was the primary subgroup detected in second-generation Japanese-Americans and in southern Californians; however, the Ic subgroup prevalent in native Japanese was rare in these populations. Since the European subgroup (Ib-2) predominated in the studied geographic area, the findings demonstrate that transmission outside the family is common in the spread of BKV, unlike previous findings for JC polyomavirus.     

Relationships between BK virus lineages and human populations

BK polyomavirus (BKV) is ubiquitous in human populations, infecting children asymptomatically and then persisting in the kidney, in which it can cause nephropathy in renal transplant patients. BKV isolates are classified into four subtypes (I-IV) using serological or genotyping methods, and subtype I is further divided into four subgroups, Ia, Ib-1, Ib-2, and Ic, based on DNA sequence variations. To clarify whether there is an association between BK virus lineages and human populations, we examined BKV-positive urine samples collected from immunocompetent individuals at various locations in Europe, Africa, and Asia. Partial BKV DNA sequences (n=299) in these samples were determined and subjected to phylogenetic and single nucleotide polymorphism analysis to classify BKV isolates around the world. The validity of the classification was confirmed by analyses based on complete BKV DNA sequences. Subtype I was the major subtype throughout the studied regions, and subtype IV was prevalent only in Asia and Europe. Subtype-I subgroups showed close relationships to major geographical areas. It has recently been shown that JC virus (a human polyomavirus closely related to BKV) co-evolved with human populations, and the present study thus suggests that host-linked evolution is the general mode of polyomavirus evolution. Additionally, our results indicate certain unique aspects of the relationship between BKV and humans.     

The human polyomavirus BK: Potential role in cancer

In human cancer, a role has been suggested for the human polyomavirus BK, primarily associated with tubulointerstitial nephritis and ureteric stenosis in renal transplant recipients, and with hemorrhagic cystitis in bone marrow transplant (BMT) recipients. After the initial infection, primarily unapparent and without clinical signs, the virus disseminates and establishes a persistent infection in the urinary tract and lymphocytes. There is correlative evidence regarding potential role of polyomavirus BK in cancer. In fact, the BK virus (BKV) DNA (complete genome and/or subgenomic fragments containing the early region) is able to transform embryonic fibroblasts and cells cultured from kidney and brain of hamster, mouse, rat, rabbit, and monkey. Nevertheless, transformation of human cells by BKV is inefficient and often abortive. Evidence supporting a possible role for BKV in human cancer has accumulated slowly in recent years, after the advent of polymerase chain reaction (PCR). BKV is known to commonly establish persistent infections in people and to be excreted in the urine by individuals who are asymptomatic, complicating the evaluation of its potential role in development of human cancer. Therefore, there is no certain proof that human polyomavirus BK directly causes the cancer in humans or acts as a cofactor in the pathogenesis of some types of human cancer.     

Human polyoma virus BK DNA detection by nested PCR in renal transplant recipients

Several studies report a correlation between the human polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients in whom immunosuppressive treatment is thought to allow or induce reactivation of the virus. Furthermore, it is described that nephropathy may result from the use of newly introduced immunosuppressive drugs. In the present study, we evaluated the presence of BKV DNA by nested-PCR (n-PCR) in urine and serum samples from 35 renal transplant patients related to the immunosuppressive regimens and renal function.     

Quantitative viral load measurement for BKV infection in renal transplant recipients as a predictive tool for BKVAN

Infection by polyomavirus BK (BKV) is an emerging problem in the clinical management of renal transplant patients because it is responsible for nephropathy and consequently can cause loss of the transplanted organ (BKV associated nephropathy, BKVAN). Aim of this study was to evaluate the use of blood viral load measurement as a screening tool for diagnosis of BKV infection and to identify a threshold value for the management of patients. A total of 75 kidney transplant patients, corresponding to 338 consecutive plasma samples, were analyzed by an automatic system for nucleic acid extraction and quantitative real-time polymerase chain reaction (PCR) for detection of BKV. BKV was detected in 170 samples (26 patients) with a median viral load of 4.1 log10 copies/mL; among these 26 patients, seven (34.7%) were found to have BKVAN on allograft biopsy together with a median viral load of 5 log10 copies/mL. The ROC curve analysis identified a viral load equal to 4.1 log10 copies/mL as the best discriminant cut-off value to predict the disease and to identify patients at risk of developing BKVAN.     

Occurrence of the African subgroup (Ia) of BK polyomavirus in younger Japanese children

BK polyomavirus (BKV) is ubiquitous among humans, usually infecting them asymptomatically during childhood. BKV persists in renal tissue of individuals and their progeny are excreted in urine, particularly in immunocompromised patients. JC virus, another human polyomavirus, has been considered to be transmitted from parents to children during prolonged cohabitation. However, BKV has been supposed to be transmitted not only within but also outside the family. In the present study, to clarify this possibility, we analyzed phylogenetically 35 BKV which were excreted in the urine by Japanese children and adults undergoing stem cell transplantation. Subtypes I, III and IV were detected in 15, two and one children and in 15, one and one adults, respectively. Among 15 subtype I isolates from children, three, four and eight belonged to subgroups Ia, Ib-1 and Ic, respectively. All the three children from whom Ia was detected were less than 9 years old. In contrast in the adults, three subtype I belonged to Ib-1 and the other 12 to Ic. These findings may reflect the recent transmission of BKV Ia strains to Japanese children.     

JC polyomavirus infection in candidates for kidney transplantation living in the Brazilian Amazon Region

This study evaluated the relative occurrences of BK virus (BKV) and JC virus (JCV) infections in patients with chronic kidney disease (CKD). Urine samples were analysed from CKD patients and from 99 patients without CKD as a control. A total of 100 urine samples were analysed from the experimental (CKD patients) group and 99 from the control group. Following DNA extraction, polymerase chain reaction (PCR) was used to amplify a 173 bp region of the gene encoding the T antigen of the BKV and JCV. JCV and BKV infections were differentiated based on the enzymatic digestion of the amplified products using BamHI endonuclease. The results indicated that none of the patients in either group was infected with the BKV, whereas 11.1% (11/99) of the control group subjects and 4% (4/100) of the kidney patients were infected with the JCV. High levels of urea in the excreted urine, low urinary cellularity, reduced bladder washout and a delay in analysing the samples may have contributed to the low prevalence of infection. The results indicate that there is a need to increase the sensitivity of assays used to detect viruses in patients with CDK, especially given that polyomavirus infections, especially BKV, can lead to a loss of kidney function following transplantation.     

Expression of viral early functions in rat 3Y1 cells infected with human papovavirus BK.

A plaque morphology mutant (pm-522) of BK virus (BKV) with a small deletion at map unit 0.72 can readily transform rat 3Y1 cells, but wild-type BKV (wt-501) cannot. We examined the expression of the viral early functions in BKV (wt-501 or pm-522)-infected 3Y1 cells within a 2-week period after infection, before foci of transformed cells became detectable, to know how the difference between the two BKVs occurs. After a high-multiplicity infection, comparable amounts of free viral DNA (forms I and II) were found by Southern blotting analyses to persist in the nuclei of the cells infected with wt and pm BKVs. Whereas the proportion of T antigen-positive cells, as revealed by the indirect immunofluorescence method with complement, remained at a level of 60% in pm BKV infection, the level of T antigen-positive cells in wt BKV infection decreased from the initial 45% to 1% on day 9. The results obtained by the immunoprecipitation analyses of radiolabeled proteins from the infected cells were consistent with the immunofluorescence data. Viral early mRNA was detectable on day 2 and increased on day 9 in pm BKV infection, but in wt BKV infection, the low level of early mRNA detected on day 2 disappeared on day 9. Cell DNA synthesis and cell growth were enhanced more in pm BKV infection than in wt BKV infection. The low level of viral DNA synthesis that occurred in the infected rat cells was more prominent in pm BKV infection than in wt BKV infection. These data indicate that the expression of viral early functions continued much longer in pm BKV-infected rat cells than in wt BKV-infected rat cells, where the expression was probably repressed soon after infection. Continued T antigen production directed by the unintegrated viral genomes appears to be required for efficient transformation of rat cells by BKV.     

Interplay of cellular and humoral immune responses against BK virus in kidney transplant recipients with polyomavirus nephropathy

Reactivation of the polyomavirus BK (BKV) causes polyomavirus nephropathy (PVN) in kidney transplant (KTx) recipients and may lead to loss of the renal allograft. We have identified two HLA-A*0201-restricted nine-amino-acid cytotoxic T lymphocyte (CTL) epitopes of the BKV major capsid protein VP1, VP1(p44), and VP1(p108). Using tetramer staining assays, we showed that these epitopes were recognized by CTLs in 8 of 10 (VP1(p44)) and 5 of 10 (VP1(p108)) HLA-A*0201+ healthy individuals, while both epitopes elicited a CTL response in 10 of 10 KTx recipients with biopsy-proven PVN, although at variable levels. After in vitro stimulation with the respective peptides, CTLs directed against VP1(p44) were more abundant than against VP1(p108) in most healthy individuals, while the converse was true in KTx recipients with PVN, suggesting a shift in epitope immunodominance in the setting of active BKV infection. A strong CTL response in KTx recipients with PVN appeared to be associated with decreased BK viral load in blood and urine and low anti-BKV antibody titers, while a low or undetectable CTL response correlated with viral persistence and high anti-BKV antibody titers. These results suggest that this cellular immune response is present in most BKV-seropositive healthy individuals and plays an important role in the containment of BKV in KTx recipients with PVN. Interestingly, the BKV CTL epitopes bear striking homology with the recently described CTL epitopes of the other human polyomavirus JC (JCV), JCV VP1(p36) and VP1(p100). A high degree of epitope cross-recognition was present between BKV and corresponding JCV-specific CTLs, which indicates that the same population of cells is functionally effective against these two closely related viruses.     

Human polyomaviruses JC and BK in the urine of Brazilian children and adolescents vertically infected by HIV

The aim of this study was to characterize the urinary excretion of the BK (BKV) and JC (JCV) human polyomaviruses in a cohort of human immunodeficiency virus (HIV)-infected children and adolescents. One hundred and fifty-six patients were enrolled: Group I included 116 HIV-infected children and adolescents [median age = 11.4 years (y); range 1-22 y]; Group II included 40 non-HIV-infected healthy controls (median age = 11.37 y; range 7-16 y). Single urine samples from both groups were screened for the presence of JCV and BKV DNA by polymerase chain reaction at enrolment. The overall rate of JCV and BKV urinary excretion was found to be 24.4% and 40.4%, respectively (n = 156). Group I had urinary excretion of JCV and BKV in 27.6% and 54.3% of subjects, respectively. In contrast, Group II showed positive results for JCV in 17.5% of subjects and for BKV in 12.5% of subjects (p Pearson JCV = 0.20; p Pearson BKV < 0.0001). In Group I, there was no association between JCV/BKV shedding and age, gender or CD4 values. Patients with an HIV viral load < 50 copies/mL had a lower excretion of BKV (p < 0.001) and a trend of lower JCV excretion (p = 0.07). One patient in Group I (1/116, 0.9%) showed clinical and radiological features consistent with progressive multifocal leukoencephalopathy, suggesting that children with HIV/polyomavirus coinfection should be kept under surveillance.     

Real-Time RT-PCR Assay for the Quantitation of Polyomavirus BK VP1 mRNA Levels in Urine

In renal transplant recipients, polyomavirus BK can reactivate resulting in graft nephropathy. Screening for BK virus replication may allow for earlier interventions with reduced allograft loss. The measurement of urinary cell BKV VP1 mRNA for identify viral replication levels at risk of developing nephropathy has been proposed. In this article, the development, optimization, and standardization of a Taqman real-time RT-PCR assay for the quantitation of BKV VP1 mRNA levels in urine is described. Subsequently, the method has been validated on urine specimens obtained from renal transplant recipients. The use of VP1 mRNA measurement as a marker for viral replication and a tool for noninvasive diagnosis of nephropathy should be regarded with great caution, given the potentially limited positive predictive value and the drawbacks associated with the complexity of the real-time RT-PCR assay requiring an expert well trained operator and the relatively poor cost-efficiency ratio.     

Breast cancer biomarkers predict weight loss after gastric bypass surgery

Background

Infections with polyomavirus BK virus (BKV) are a common cause of renal dysfunction after renal transplantation and may also be harmful in surgical patients with shock. The aim of the present study was to determine the frequency of BKV viremia in critically ill surgical patients with septic or hemorrhagic shock, and, if viremia is detectable, whether viremia may be associated with renal dysfunction.

Findings

A total of 125 plasma samples from 44 critically ill surgical patients with septic or hemorrhagic shock were tested by real-time polymerase chain reaction (PCR) for BKV DNA during their stay on the intensive care unit (ICU). BKV viremia occurred in four patients, i.e. in three of the septic and in one of the hemorrhagic shock group. There was no association between viremia and renal dysfunction. All positive samples contained a low viral load (< 500 copies/ml).

Conclusions

Since BK viremia was rarely found and with low viral load only in critically ill surgical patients with shock, it is very unlikely that BK viremia results in BK nephropathy later on.     

Early Events during BK Virus Entry and Disassembly

BK virus (BKV) is a nonenveloped, ubiquitous human polyomavirus that establishes a persistent infection in healthy individuals. It can be reactivated, however, in immunosuppressed patients and cause severe diseases, including polyomavirus nephropathy. The entry and disassembly mechanisms of BKV are not well defined. In this report, we characterized several early events during BKV infection in primary human renal proximal tubule epithelial (RPTE) cells, which are natural host cells for BKV. Our results demonstrate that BKV infection in RPTE cells involves an acidic environment relatively early during entry, followed by transport along the microtubule network to reach the endoplasmic reticulum (ER). A distinct disulfide bond isomerization and cleavage pattern of the major capsid protein VP1 was observed, which was also influenced by alterations in pH and disruption of trafficking to the ER. A dominant negative form of Derlin-1, an ER protein required for retro-translocation of certain misfolded proteins, inhibited BKV infection. Consistent with this, we detected an interaction between Derlin-1 and VP1. Finally, we show that proteasome function is also linked to BKV infection and capsid rearrangement. These results indicate that BKV early entry and disassembly are highly regulated processes involving multiple cellular components.     

The kinetics of urinary shedding of BK virus in children with renal disease

Children with renal diseases are typically treated with immunosuppressive drugs, which place them at high risk of reactivation of the BK virus (BKV). Currently, little is known about the impact of immunosuppressive drugs on the kinetics of urinary shedding of BKV and viral reactivation in pediatric patients with renal diseases. Urine samples were collected monthly for 1 year from 20 children (median age, 9 years; range, 4–15 years) with renal diseases and subjected to real‐time PCR. Urinary shedding of BKV was detected in 35% (7/20) of the patients, three of these patients having persistent viral DNA excretion (two cases, twelve times; one case, four times) and four having intermittent viral DNA excretion. Thirty‐four of the 240 urine samples contained BKV DNA (median copy numbers, 5.66 log copies/mL; range, 2.45–7.69 log copies/mL). In two of the cases with persistent viral shedding, high copy numbers (range, 4.57–7.69 log copies/mL) of BKV DNA were detected in all 12 urine samples collected. In the other case with persistent viral excretion, a range of 2.45–3.98 log copies/mL of BKV DNA was detected in the four urine samples collected between the 9th and 12th sampling time points. Additionally, high copy numbers (range, 3.12–4.36 log copies/mL) of BKV DNA were detected intermittently in the urine samples of the other four cases. No remarkable correlations were found between the kinetics of BKV DNA loads and urinary findings such as proteinuria and hematuria. The present data demonstrate the kinetics of urinary BKV shedding in pediatric patients with renal diseases. Additionally, no pathogenic role for BKV infection was identified.     

Detection of BK virus in urine from renal transplant subjects by mass spectrometry

Background

The diagnosis and management of BK virus (BKV) reactivation following renal transplantation continues to be a significant clinical problem. Following reactivation of latent virus, impaired cellular immunity enables sustained viral replication to occur in urothelial cells, which potentially leads to the development of BKV-associated nephropathy (BKVAN). Current guidelines recommend regular surveillance for BKV reactivation through the detection of infected urothelial cells in urine (decoy cells) or viral nucleic acid in urine or blood. However, these methods have variable sensitivity and cannot routinely distinguish between different viral subtypes. We therefore asked whether mass spectrometry might be able to overcome these limitations and provide an additional non-invasive technique for the surveillance of BKV and identification of recipients at increased risk of BKVAN.

Results

Here we describe a mass spectrometry (MS)-based method for the detection of BKV derived proteins directly isolated from clinical urine samples. Peptides detected by MS derived from Viral Protein 1 (VP1) allowed differentiation between subtypes I and IV. Using this approach, we observed an association between higher decoy cell numbers and the presence of the VP1 subtype Ib-2 in urine samples derived from a cohort of 20 renal transplant recipients, consistent with the hypothesis that certain viral subtypes may be associated with more severe BKVAN.

Conclusions

This is the first study to identify BK virus proteins in clinical samples by MS and that this approach makes it possible to distinguish between different viral subtypes. Further studies are required to establish whether this information could lead to stratification of patients at risk of BKVAN, facilitate distinction between BKVAN and acute rejection (AR), and ultimately improve patient treatment and outcomes.