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Table of Contents
REVIEW ARTICLE
Year : 2018  |  Volume : 12  |  Issue : 1  |  Page : 7-12

Role of urinary viral haufen in diagnosis of polyomavirus nephropathy: A systematic review


1 Department of Pathology, ESIC Model Hospital, Sector-9, Gurugram, Haryana, India
2 Department of Pediatrics, All Institute of Medical Sciences, New Delhi, India

Date of Web Publication29-Mar-2018

Correspondence Address:
Dr. Anju Khairwa
Department of Pathology, ESIC Model Hospital, Sector-9, Gurugram, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijot.ijot_68_17

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  Abstract 

Renal biopsy is a gold standard method for diagnosis of BK polyomavirus nephropathy (PVN); however, it is an invasive procedure, and it can be false negative due to sampling error. Many noninvasive screening strategies such as decoy cell in urine and viral load assessments using polymerase chain reaction in urine and in plasma have a limited role in diagnose of PVN. Recently, urinary viral Haufen (UVH) is found to be very useful for diagnosis of PVN. The aim of this study was to evaluate the efficacy of UVH in the diagnosis of PVN in all transplant patients. In this systematic review, we included studies where UVH was used for diagnosis of PVN. We searched PubMed, EMBASE, Google Scholar, and Cochrane database of systematic reviews. Two authors independently selected studies and extracted data for review. Five studies were included in this study. One study reported sensitivity, specificity, positive predictive value, and negative predictive values of UVH against kidney biopsy as 100%, 99%, 97%, and 100%, respectively, for diagnosis of PVN in all type of transplant patients. Second study described technique of UVH estimation. Two studies correlated UVH with severity of PVN. Fifth study demonstrated that UVH formation occurs in the setting of high concentration of Tamm–Horsfall protein in urine and also reported UVH as a marker of PVN in mice. Data were not appropriate for meta-analysis. A limited number of studies showed that UVH may be a novel noninvasive biomarker to diagnosis and to predict the severity of PVN in transplant patients.

Keywords: Haufen, polyomavirus nephropathy, systematic, transplant, urinary viral Haufen


How to cite this article:
Khairwa A, Jat KR. Role of urinary viral haufen in diagnosis of polyomavirus nephropathy: A systematic review. Indian J Transplant 2018;12:7-12

How to cite this URL:
Khairwa A, Jat KR. Role of urinary viral haufen in diagnosis of polyomavirus nephropathy: A systematic review. Indian J Transplant [serial online] 2018 [cited 2018 Aug 21];12:7-12. Available from: http://www.ijtonline.in/text.asp?2018/12/1/7/228934




  Introduction Top


Polyomavirus nephropathy (PVN) is a common infectious complication after renal transplantation. The most common etiological agent for PVN is BK virus.[1] Overall incidence of PVN in kidney transplant recipients ranges from 4% to 12%.[2],[3],[4],[5] The confirmatory gold standard method for diagnosis PVN is renal biopsy.[6],[7] However, it is an invasive procedure, and sometime, it can be false negative due to sampling error.[8]

Many noninvasive screening strategies have been introduced to assess the risk for PVN. It can be diagnosed by demonstrating decoy cell shedding in urine and by viral load assessments using polymerase chain reaction (PCR) in urine (BK viruria) and in plasma (BK viremia).[9],[10] These screening methods have limited predictive value to diagnose PVN as they are not “kidney disease-specific” and cannot reliably distinguish between clinically insignificant polyomavirus replication and manifest intrarenal disease (BK viral nephropathy).[11],[12] To overcome these limitations, a new diagnostic method is described recently. The urinary viral Haufen (UVH) test is a novel noninvasive assay to predict PVN accurately.[13] UVH is defined as cast-like, three-dimensional polyomavirus aggregates, that is, “Haufen” (German for heap or stalk) in voided urine samples detected by negative staining electron microscopy. Furthermore, in contrast to commonly used laboratory tests such as quantitative PCR, UVH testing can detect intrarenal polyomavirus burden and severity of PVN and can identify early disease stages.[14] The test has positive and negative predictive values (PPV and NPV) of >90%, for diagnosis of PVN, far exceeding any other currently used screening assay.[1] Detection of UVH can be used to diagnose BKN accurately and noninvasively with good sensitivity and specificity. The UVH may useful to diagnose PVN noninvasively. Therefore, we plan this systematic review to evaluate the efficacy of UVH test in the diagnosis of PVN in all transplant patients.


  Methods Top


In this systematic review, we included studies where UVH method was used in the diagnosis of PVN in transplant patients. Studies including both children and adults were eligible for inclusion in review. We included all posttransplant patients including kidney transplant. Renal biopsy was considered as reference gold standard method for the diagnosis of PVN. We searched PubMed (inception to July 31th, 2017), EMBASE (inception to July 31th, 2017), Google scholar (inception to July 31th, 2017), and Cochrane Database Reviews, July 2017. Search strategy for PubMed included “Haufen” OR Haufen PVN AND urinary Haufen PVN). We searched EMBASE with key words “Haufen”/exp OR PVN AND (“Haufen”/exp OR urinary Haufen PVN). In Google Scholar, we searched “urinary Haufen/Haufen PVN.” References of included studies were hand searched for additional studies. Two authors independently selected studies and extracted data for review.


  Results Top


PubMed, EMBASE, Google Scholar, and Cochrane library search revealed six, 14, four, and zero results, respectively. After excluding duplicates and irrelevant studies, five studies were included in the review.[1],[13],[14],[16] The study selection flow is shown in [Flow Chart 1]. The extracted data from included studies are shown in [Table 1].
Table 1: Data extraction from included studies

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Singh et al. evaluated Haufen in 194 urine samples from 139 control patients and in 143 samples from 21 patients with BK PVN using negative-staining electron microscopy.[1] Authors reported sensitivity, specificity, PPV, and NPV of UVH against kidney biopsy as 100%, 99%, 97%, and 100%, respectively for diagnosis of PVN in all type of transplant patients. The comparison between UVH and other screening method of PVN had shown in [Table 1]. The Haufen in urine correlated tightly with biopsy-confirmed BK PVN (concordance rate 99%).[1] Sing et al. found that all controls were Haufen-negative; however, high viremia and viruria were detected in 8% and 41% of control samples, respectively. k statistics showed fair to good agreement of viruria and viremia with BK PVN or with Haufen shedding and demonstrated an excellent agreement between Haufen and PVN (κ = 0.98).[1] UVH and decoy cells had shown [Figure 1]a and [Figure 1]b.
Figure 1: (a) Polymavirus Haufen has shown in negative staining electron microscopy on a voided urine sample from a patient with PVN.[22] Large three-dimensional cast-like viral aggregates, the so-called polyomavirus-Haufen. (Electron microscopy × 80,000),(b) Decoy cells (Polyomavirus inclusion in cells) in Liquid-based urine cytology preparation. Decoy cells are large and having glassy-appearing intranuclear viral inclusion bodies.[22] (Papanicolaou stain, 600× original magnification). Reproduced from reference no. 22 with permission of publisher and author of Heptinstall's Pathology of the Kidney, 7th Edition

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Singh et al. studied more than 400 urine samples from over 180 patients and developed easy-to-follow protocols to detect Haufen that included optimal specimen preservation and preparation, sample clarification, and concentration.[13] They provided detailed methods to detect Haufen and illustrate pitfalls, including “Haufen-look-alikes,” which can hamper the interpretation. They described UVH detection by negative staining electron microscopy.[13] The demonstration of urinary Haufen in voided urine is a new urinary biomarker with positive and NPV of >95%.[13]

Singh et al. counted polyomavirus-Haufen in 40 urine samples from patients with biopsy-proven definitive PVN.[14] The number of PV-Haufen was correlated with both histologic PVN disease Grades 1–3 and the number of SV40-T–expressing cells as indicators of intrarenal PV replication in corresponding renal allograft biopsies. Singh et al. also reported PV-Haufen counts showed excellent correlation (α0.77–0.86) with severity of intrarenal PV replication and disease grades.[14] Authors also compared quantitative PV-Haufen analyses and BK viremia (quantitative PCR) and BK viruria (quantitative PCR and decoy cell counts). Low PV-Haufen numbers strongly correlated with early PVN Grade 1 and minimal intrarenal expression of SV40-T antigen (P< 0.001). In comparison, BK viremia and viruria levels by PCR showed only modest correlations with histologic SV40-T expression (α0.40–0.49)and showed no significant correlation with disease grades or minimal intrarenal PV replication.[14],[15] There was any correlations between PVN severity and urinary decoy cell counts. In contrast to conventional quantitative PCR assays or decoy cell counts, quantitative UVH testing accurately reflected the severity of PV replication, tissue injury, and PVN disease grades.[14]

Laskin et al. evaluated polyoma-BK virus associated with hemorrhagic cystitis and also with PVN after hematopoietic cell transplantation (HCT). He examined 21 suitable urine samples from 14 patients, who were selected from repositories for having varying degrees of BK viremia (range, 0–1.0 × 108 copies/mL), hemorrhagic cystitis (present/absent), and data on kidney function. The UVH were detected in 5 of 14 patients (35.7%) and 7 of 21 (33.3%) urine samples, with histologic confirmation of PVN in 1 autopsy specimen.[16] After a median of 285 days post-HCT, patients with UVH had an increased risk of dialysis-dependent renal failure (P< 0.05). All three dialysis-dependent patients had PV-Haufen and died. The presence of urinary PV-Haufen was not significantly correlated with hemorrhagic cystitis. From the 16 urines collected during BK viremia, 43.8% were PV-Haufen–positive, and 56.2% were negative.[16] The PV-Haufen was not present in the 5 urines from patients without concomitant BK-viremia. He concluded that detection of PV-Haufen suggests underlying PVN with an increased risk of kidney failure and dialysis.[16]

Nickeleit et al. demonstrated that PV-(aggregation) Haufen formation depends on a high concentration of Tamm–Horsfall protein (THP) that is present only in primary urine found in injured renal tubules.[17] THP was not detected in secreted secondary urine.[17] PV aggregation and PV-Haufen formation are THP dose-dependent and only occurs with high THP concentrations of >1 mg THP/mL fluid mimicking primary urine in injured tubules. Author found mouse model of PVN showed urinary PV-Haufen in mice with PVN and absent in control animals without PVN.[17] Thus, presence of PV-Haufen in voided urines is specific for intrarenal disease.


  Discussion Top


We evaluated the role of UVH in the diagnosis of PVN in posttransplant patients in this systematic review. The detection of PV – Haufen in voided urine samples by negative staining EM with had high interobserver agreement/reproducibility. There is new technique for detection of PV-Haufen that differs from previous studies focusing on polyomavirus infections and the urinary shedding of free, and nonclustered viruses.[12] Singh et al. described the technical details of negative staining electron microscopy on voided urine samples to facilitate the detection of Haufen.[13] Haufen is defined as singly scattered cast like virions of polyomaviruses that are three-dimensional, polyomavirus aggregates.[1] UVH is described for the first time as reliable markers of BK nephropathy.[1] UVH had excellent correlation with BK – nephropathy stages (A to C). Sing et al. showed an excellent level of agreement with a κ = of 0.98 and a nearly perfect concordance rate of 99% in BKN and Haufen.[1] The qualitative detection of Haufen had PPV and NPV of 97% and 100%, respectively, for identifying biopsy-proven BKN.[1] The qualitative detection of PV-Haufen that is present or absent in a fixed urine specimen predicted PVN with positive and NPV >95%.[14]

Sing et al. correlated the number of PV-Haufen in voided urine samples with the severity of PVN and compared the data to standard PV-screening assays. The quantitative measurement of UVH testing most accurately reflected the severity of intrarenal PV replication and virus-induced renal injury.[14],[15] The quantitative UVH testing also correlated with PVN disease grades as proposed by the Banff Working Group on PVN.[18] In early stage-A with mild PVN disease grade 1 was reflected by low numbers of urinary PV-Haufen which differed from much higher counts in the stage -B with florid PVN disease grade 2, and stage-C with late and sclerosed PVN disease grade 3. Intrarenal PV replication correlated the Haufen by counting the number of SV40 in renal biopsy which was increased in increments parallel to the increase of intrarenal PV replication and high numbers of urinary PV-Haufen reflected marked virus-induced tubular injury, a high intrarenal PV burden (correlation coefficient, 0.86).[14] Other markers which indicates intrarenal PV replication are intratubular cellular casts that contains high concentrations of uromodulin (THP). High concentrations of THP facilitate the aggregation of PV in vitro.[19] Singh et al. did not find significant correlations between quantitative PCR test results of viremia and viruria and decoy cell counts with PVN disease grades or limited PVN with only minimal intrarenal disease.[14] The quantitative urinary PV-Haufen testing could be used to monitor PVN progression or regression during persistent disease. In transplantation kidney, urinary PV-Haufen shedding follows a dynamic pattern closely reflecting the course of PVN.[1] The proof-of-concept study by Laskin et al. showed that urinary PV-Haufen testing post-HCT findings were very similar to those previously described after renal transplantation.[1],[14],[16] Laskin et al. found that PVN, based on the presence of PV-Haufen in the urine, was more common after HCT than previously thought, affecting one third of their study cohort.[16] He also found urinary PV-Haufen were present in only a subgroup of patients with BK-viremia, had evidence of chronic kidney disease, an increased risk for dialysis-dependent renal failure and not associated with hemorrhagic cystitis, which was different from other studies.[16] Sing et al. showed associations between urinary PV-Haufen shedding and renal histology in renal transplant recipients. This association is lacking in study by Laskin et al. due to the nature of underlying disease preventing invasive diagnostic interventions and collection of biopsies.[14],[16] However, their results supported a novel conceptual model for clinical testing and diagnosing disease associated with BK virus infection in HCT recipients.[16]

Nickeleit et al. also agreed the previous studies and demonstrated PV-aggregation and Haufen formation depend on a high concentration of THP in primary urine.[17] Author also reported that urinary PV-Haufen in a mouse model of PVN but not in control animals.[17]

Screening using urinary Haufen by EM is not a mass screening assay but rather a targeted clinical test. The authors recommended use of UVH testing in patients with signs of polyomavirus replication such as high BK viruria (shedding of 10 or more decoy cells or quantitative PCR test results >107 BK virus gene copies/mL urine) or any degree of BK viremia.[1] Detection of Haufen in urine can amend renal allograft biopsy interpretations, whenever biopsies are small and BKN on histologic signs of might be remain unsampled.[20],[21],[22]


  Conclusion Top


Urinary Haufen detection diagnoses BKN accurately and noninvasively with good sensitivity and specificity. The quantitative estimation PV-Haufen testing in urine can be used as a novel noninvasive approach to patient management for the diagnosis and prediction of PVN disease grades and monitoring of disease course during therapy. Larger, prospective studies, and systematically collecting urine samples at defined time points are required in the future to robust these findings.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Singh HK, Andreoni KA, Madden V, True K, Detwiler R, Weck K, et al. Presence of Urinary Haufen Accurately Predicts Polyomavirus Nephropathy. J Am Soc Nephrol 2009;20:416-27.  Back to cited text no. 1
    
2.
Benavides CA, Pollard VB, Mauiyyedi S, Podder H, Knight R, Kahan BD, et al. BK virus-associated nephropathy in sirolimus-treated renal transplant patients: Incidence, course, and clinical outcomes. Transplantation 2007;84:83.  Back to cited text no. 2
    
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Hirsch HH, Randhawa P. BK polyomavirus in solid organ transplantation. Am J Transplant 2013;13(Suppl 4):179.  Back to cited text no. 3
    
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van Aalderen MC, Heutinck KM, Huisman C, ten Berge IJ. BK virus infection in transplant recipients: clinical manifestations, treatment options and the immune response. Neth J Med 2012;70:172.  Back to cited text no. 4
    
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Ranzi AD, Prolla JC, Keitel E, Brackmann R, Kist R, dos Santos G, et al. The role of urine cytology for 'decoy cells' as a screening tool in renal transplant recipients. Acta Cytol 2012;56:543.  Back to cited text no. 5
    
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Nickeleit V, Hirsch HH, Zeiler M, Gudat F, Prince O, Thiel G, et al. BK-virus nephropathy in renal transplants-tubular necrosis, MHC-class II expression and rejection in a puzzling game. Nephrol Dial Transplant 2000;15:324–332.  Back to cited text no. 6
    
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Hirsch HH, Brennan DC, Drachenberg CB, et al. Polyomavirus associated nephropathy in renal transplantation: Interdisciplinary analyses and recommendations. Transplantation 2005;79:1277-128.  Back to cited text no. 7
    
8.
Colvin RB, Nickeleit V. Renal transplant pathology. In: Jennette JC, Olson JL, Schwartz MM, Silva FG, eds. Pathology of the Kidney, 6th ed. Philadelphia: Lippincott Williams and Wilkins; 2007. p. 1347-490.  Back to cited text no. 8
    
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Drachenberg RC, Drachenberg CB, Papadimitriou JC, Ramos E, Fink JC, Wali R, et al. Morphological spectrum of polyoma virus disease in renal allografts: Diagnostic accuracy of urine cytology. Am J Transplant 2001;1:373-81.  Back to cited text no. 9
    
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Randhawa P, Ho A, Shapiro R, Vats A, Swalsky P, Finkelstein S, et al. Correlates of quantitative measurement of BK polyomavirus (BKV) DNA with clinical course of BKV infection in renal transplant patients. J Clin Microbiol 2004;42:1176-80.  Back to cited text no. 10
    
11.
Sundsfjord A, Flaegstad T, Flø R, Spein AR, Pedersen M, Permin H, et al. BK and JC viruses in human immunodeficiencyvirus type 1-infected persons: Prevalence, excretion, viremia, and viral regulatory regions. J Infect Dis 1994;169:485-90.  Back to cited text no. 11
    
12.
Singh HK, Madden V, Shen YJ, Thompson BD, Nickeleit V. Negativestaining electron microscopy of the urine for the detection of Polyoma virus infections. Ultrastruct Pathol 2006;30:329-38.  Back to cited text no. 12
    
13.
Singh HK, Donna Thompson B, Nickeleit V. Viral Haufen are urinary biomarkers of polyomavirus nephropathy: New diagnostic strategies utilizing negative staining electron microscopy. Ultrastruct Pathol 2009;33:222-35.  Back to cited text no. 13
    
14.
Singh H, Kozlowski T, True K, Derebail V, Gasim A, Nickeleit V. Urinary polyomavirus-Haufen shedding accurately reflects intrarenal burden of polyomavirus nephropathy (PVN): Comparative quantitative analyses of different screening techniques. Modern Pathology 2011;24 (Suppl 1):1492A.  Back to cited text no. 14
    
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Whiting PF, Rutjes AWS, Westwood ME, Mallett S, Deeks JJ, Reitsma JB, et al. QUADAS-2: A revised tool for the quality assessment of diagnostic accuracy studies. Ann Intern Med 2011;155:529-36.  Back to cited text no. 15
    
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Laskin BL, Singh HK, Beier UH, Moatz T, Furth SL, Bunin N, et al. The noninvasive urinary polyomavirus haufen test predicts BK virus nephropathy in children after hematopoietic cell transplantation: A pilot study. Transplantation 2016;100:e81-7.   Back to cited text no. 16
    
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Nickeleit V. Brylawski B. Singh H. Rivier L. Urinary polyomavirus-haufen shedding in mouse and man: A proof-of-concept study for a non-invasive urine biomarker for polyomavirus nephropathy. Laboratory Investigation 2013;93 Suppl 1:390A-1A.  Back to cited text no. 17
    
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Nickeleit V, Brylawski B, Singh HK, Rivier L. Urinary polyomavirus-Haufen shedding in mouse and man: A proof-of-concept study for a non-invasiveurine biomarker for polyomavirus nephropathy. Lab Invest 2013;93:390A.  Back to cited text no. 19
    
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Nickeleit V, Singh HK, Mihatsch MJ. Polyomavirus nephropathy: Morphology, pathophysiology, and clinical management. Curr Opin Nephrol Hy 2003;12:599-605.  Back to cited text no. 21
    
22.
Nickeleit V, Mengel M, Colvin RB. Renal Transplant Pathology. In: Jennette JC, Olson JL, Silva FG, Vivette D. D'Agati, editors. Heptinstall's Pathology of the Kidney. 7th ed. Wolters Kluwer; 2015. p.1321-431.  Back to cited text no. 22
    


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