Parvovirus B19 infection and renal injury: report of 4 cases and analysis of immunization and viremia in an adult cohort of 100 patients undergoing a renal biopsy.

BACKGROUND: The seroprevalence of human Parvovirus B19 (PVB19) is 70-85 % in adults worldwide. PVB19 is the etiologic agent of the fifth disease, is a cause of aplastic anemia, and can be associated with renal injury. We aimed to describe the cases of 4 patients with renal injury related to PVB19 primary infection, and to evaluate the seroprevalence of PVB19 and the incidence of PVB19 primary infection in patients undergoing a native kidney biopsy. METHODS: Cases of PVB19 infection with renal injury were reviewed from the archives of the department of Nephrology. A systematic screening of anti-PVB19 IgG and IgM antibodies and viral DNA was performed in sera from 100 consecutive patients with a renal biopsy in 2017-2018. RESULTS: The 4 patients with PVB19 infection-associated renal disease displayed: one lupus-like glomerulonephritis (GN) without lupus auto-antibodies, one minimal change disease with tubular necrosis, one secondary hemolytic and uremic syndrome and one membrano-proliferative GN. In the 100 patients biopsied, 67 had elevated anti-PVB19 IgG, among whom 8 had elevated IgM, without circulating viral DNA, without any particular renal pathological pattern. One additional patient showed a seroconversion at the time of renal biopsy, which revealed a class V lupus nephritis. CONCLUSION: PVB19 primary infection can be associated with different renal diseases. The seroprevalence of PVB19 among patients with a renal biopsy is similar to the overall population, and primary infection is rarely documented (1%) after systematic screening. Whether PV19 is nephrotoxic, or triggers renal endothelial injury and immune activation, remains to be elucidated. Abstract BACKGROUND: The seroprevalence of human Parvovirus B19 (PVB19) is 70-85 % in adults worldwide. PVB19 is the etiologic agent of the fifth disease, is a cause of aplastic anemia, and can be associated with renal injury. We aimed to describe the cases of 4 patients with renal injury related to PVB19 primary infection, and to evaluate the seroprevalence DNA was performed in sera from 100 consecutive patients with a renal biopsy in 2017-2018. RESULTS: The 4 patients with PVB19 infection-associated renal disease displayed: one lupus-like glomerulonephritis (GN) without lupus auto-antibodies, one minimal change disease with tubular necrosis, one secondary hemolytic and uremic syndrome and one membrano-proliferative GN. In the 100 patients biopsied, 67 had elevated anti-PVB19 IgG, among whom 8 had elevated IgM, without circulating viral DNA, without any particular renal pathological pattern. One additional patient showed a seroconversion at the time of renal biopsy, which revealed a class V lupus nephritis. CONCLUSION: PVB19 primary infection can be associated with different renal diseases. The seroprevalence of PVB19 among patients with a renal biopsy is similar to the overall population, and primary infection is rarely documented (1%) after systematic screening. Whether PV19 is nephrotoxic, or triggers renal endothelial injury and immune activation, remains to be elucidated.


Introduction
Human parvovirus B19 (PVB19) is a ubiquitous small ssDNA virus, known as the etiologic agent of the fifth disease. Most adults worldwide show evidence of past infection (between 70 and 85%), but a primary infection can occur lately (1). Infectivity shows seasonal variation, and is more common in spring (2).
In nephrology, PVB19 infection is a matter of concern mainly in kidney transplant recipients, as a cause of aplastic anemia and pure red cell aplasia. The incidence of PVB19 infection after kidney transplantation either as a primary infection or a reactivation, varies between 2 and 30 % (3). PVB19 has also been described as a possible cause of renal injury. Several cases of glomerulonephritis (GN) occurring after a PVB19 primo-infection have been reported in the literature, although the pathogenic role of PVB19 was difficult to establish. Renal presentation was mostly post-infectious GN, but collapsing focal segmental glomerulosclerosis (FSGS), membrano-proliferative GN, and thrombotic microangiopathy have also been reported. (4)(5)(6)(7)(8) Extra-hematological and extra-renal signs can vary from mild or moderate (rash, symmetric arthralgia or arthritis) to severe manifestations (myocarditis, pericarditis, cryoglobulinemic vasculitis, lymphoproliferation), depending on the age, comorbidity, and immunological status of the host (9).
The aims of this study were: 1) to describe the presentations and outcomes of 4 patients who presented with a renal disease following a primary infection by PVB19 in the department of Nephrology of our University hospital (Hôpital de la Conception, Marseille, France); 2) to evaluate, by a systematic screening, the seroprevalence of PVB19 and the incidence of PVB19 primary infection in a cohort of consecutive patients who underwent a native kidney biopsy in our department.
anti-dsDNA antibodies. Bone marrow aspiration was normal, and abdominal CT scanner revealed no abnormality.
The renal biopsy showed minimal change disease and acute tubular necrosis, without immune deposits or glomerular proliferation. Viral and bacterial serologies were negative (HIV, HBV, HCV, Treponema pallidum), except for PVB19 which showed elevated titers of both IgG and IgM, with a positive viremia. PVB19 was also detected by PCR in the renal tissue.
Case report n°3: Hemolytic and Uremic Syndrome (HUS) A 28-year-old man was admitted to the Nephrology Intensive Care Unit in 2012 for HUS. He reported fever, abdominal pain and non-hemorrhagic diarrhea, 10 days earlier. He came to the emergency room for a worsening asthenia. Physical examination showed only pallor, a blood pressure of 128/97 mmHg, without fever. The blood tests revealed acute renal failure (serum creatinine 6.14 mg/dl), mechanical hemolytic anemia (Hemoglobin 11.9 g/dL, undetectable serum haptoglobin, positive schizocytes 1.5%, elevated lactate deshydrogenase 2000 UI/L), without regeneration (reticulocytes 4.2 G/L) associated with a thrombocytopenia (platelets 16 G/L). Urinary tests showed proteinuria (0.8 g/L) and leukocyturia (350/mm3) without hematuria.
Complement fractions were normal, and no abnormality was detected in the complement alternative pathway proteins. There was no marker of auto-immunity, and the search for E.Coli O157:H7 and Shigatoxin in the stool was negative. Because of the low reticulocyte count, despite hemolytic anemia, a PVB19 infection was suspected and confirmed, with elevated IgG and IgM titers and positive viremia.
One session of plasma exchange was performed, and the patient subsequently reached hematological remission, followed by renal remission. Nine days after his admission, platelet count had normalized (382 G/L), LDH decreased (400 UI/L), and renal function improved (serum creatinine 3.64 mg/dL). No renal biopsy was performed. The patient recovered a normal renal function (eGFR 95 ml/min/1,73m2) immunosuppressive therapy (including corticosteroids) at the time of the biopsy.
Elevated titers of anti-PVB19 IgG antibodies were found in 67 (67%) patients, among whom 8 (2 females and 6 males, aged 31 to 83 years) also had elevated IgM titers, without PVB19 viremia ( Table   2). The renal pathological results of these 8 patients with both IgG and IgM antibodies were: acute kidney tubular necrosis in 2, minimal change disease in 1, class III lupus nephritis in 1, MPGN with a "full-house" pattern of endo-membranous deposits in 1 (lupus-like GN without lupus auto-antibodies), pauci-immune crescentic GN in 1, hypertensive nephropathy in 1, acute interstitial nephritis with lymphocytic infiltration in 1.
One additional patient, a 63-year-old women, initially had elevated anti-PVB19 IgM without IgG antibodies, and subsequently developed anti-PVB19 IgG when she was tested again 4 months later.
This was consistent with a PVB19 primary infection at the time of renal biopsy, although viremia was negative. She had been admitted for arthralgia, and nephrotic syndrome with acute kidney injury (serum creatinine 1.23 mg/dl). Laboratory tests had revealed a non-regenerative anemia (hemoglobin 9,6 g/dl, reticulocytes 26 G/L). Renal pathological examination showed a membranous nephropathy ( Figure 1D). Serum anti-PLA2R antibodies were negative, but there were positive anti-nuclear (> 1/1280) and anti-dsDNA (41 UI/mL) antibodies. The diagnosis retained was a class V lupus nephritis.
Overall, only 1 patient (1%) had a documented primary PVB19 infection in this cohort, and no patient had a PVB19 viremia. Among the 9 patients with elevated IgM titers, 4 had auto-reactive antibodies (antinuclear, ANCA, anti-MBG or PLA2R antibodies), and 6 had a mixed cryoglobulinemia.

Discussion
We confirm in this work the high prevalence of PVB19 immunization in adult patients in Nephrology.
We report 4 cases of different renal diseases associated with acute or chronic PVB19 infection, ranging from immune deposition diseases to minimal change disease and thrombotic microangiopathy. Although the causality of PVB19 primary infection could not be established in these detection of viral DNA in tissues does not necessarily indicate active viral infection and must be interpreted with caution (11).
Only one additional patient with PVB19 primary infection was identified through the systematic screening of 100 consecutive patients who underwent a kidney biopsy. PVB19 infection is thus probably not a frequent and underdiagnosed cause of renal injury. However, it could be searched for in patients with unexplained lupus-like GN, MCD or TMA, especially after the onset of viral-like symptoms and in periods of seasonal epidemic. Regrettably, as opposed to other viral epidemics, such as flu or gastro-enteritis, no specific health alert is displayed for PVB19 incidence peaks, and PVB19 infection is not a notifiable disease in most countries.
In western countries, PVB19 immunization is observed in 5-15% of children aged 1-5 years, who are the main source of the virus transmission, in 50-60% of older children and young adults, and in over 85% in persons older than 70 years (12)(13)(14). Our results (67% in patients with a mean age of 51 years) are consistent with previous data. immunoglobulins has proved to be effective, but no controlled studies have been carried out (17,18).
Reduction of immunosuppressive medication, when possible, is often recommended.
The specificity and sensitivity of PVB19 specific IgM assay vary between 70 and 100%, meaning that false positive results are possible (19). Autoimmune antibodies, such as a rheumatoid factor, cryoglobulinemia or antinuclear antibodies, can cross-react with PVB19 serology and yield false positive IgM (20). Most patients with positive anti-PVB19 IgM antibodies from the present cohort had a mixed cryoglobulinemia. This could either reflect a cause of false positive serological results, or confirm the suspected role of PVB19 in the development of infectious cryoglobulinemia. Although hepatitis C virus (HCV) infection is the most frequent cause of infectious cryoglobulinemia (70-90%) (21), several case reports of PVB19-related cryoglobulinemic vasculitis have been reported (22,23). In the French nationwide CryoVas survey, among 18 patients with non-HCV-related infectious cryoglobulinemic vasculitis, one (5%) was related to PVB19 (24). A tendency for a higher seroprevalence of PVB19 was also documented among patients with cryoglobulinemia compared to a control group (64.9% versus 50%, NS) (25).
Moreover, a transient positivity of anti-neutrophil cytoplasmic antibodies (ANCA), with anti-proteinase 3 or anti-myeloperoxidase specificity, has been reported in 10% of patients during acute PVB19 infection (26), and is a potential pitfall for the diagnosis of ANCA-associated vasculitis. Recently, an association between persistent PVB19 infection and the production of antiphospholipid antibodies in pediatric and adult patients with rheumatic diseases has also been described (27). Molecular mimicry could be a major pathogenic mechanism triggering the production of auto-antibodies after PVB19 infection (28). Several reports have described lupus-like manifestations related to PVB19 infection, including some with acute glomerulonephritis, with transient positive anti-nuclear or anti-dsDNA antibodies (29). The correct diagnosis of PVB19 infection is particularly important in this setting, to avoid an inadequate and potentially harmful immunosuppressive therapy (29,30).
Although it is now established that the pathogenicity of PVB19 is not restricted to the erythroid progenitor cells, the mechanisms of PVB19-related renal lesions are still poorly understood. The proposed mechanisms include cytopathic effects on glomerular endothelial cells, glomerular deposition of immune complexes resulting from the infection, or the development of immune disorders after PVB19 infection. The direct infection of glomerular epithelial cells could be the mechanism of renal injury in cases of FSGS, including collapsing glomerulopathy (6). Indeed, PVB19 viral DNA was detected in kidney biopsies of patients with non-HIV-related collapsing FSGS, and was precisely located in visceral and parietal epithelial cells. However, viral DNA was also detected in control samples and in other glomerular lesions, which questions the specificity of this finding (31). In cases of TMA and vasculitis, the direct infection of glomerular endothelial cells has also been proposed as the trigger of endothelial activation and lesion, leading to the subsequent thrombotic and inflammatory response with complement activation (7). Indeed, the endocytosis of PVB19 by endothelial cells is enhanced by the presence of anti-PVB19 antibodies and their linkage to the C1q receptor (32). The glomerular deposition of circulating immune complexes, comprising viral antigens and host antiviral antibodies, could also lead to a post-infectious glomerulonephritis, which is the pathological lesion most frequently described in the literature. In addition, PVB19 could be implicated in the activation of complement alternative pathway (CAP), since C3 level is often decreased in PVB19 infection with kidney involvement (33). Here, the case report n°4, showing MPGN with C3 deposits following a PVB19 infection in a patient with sickle cell disease, is particularly original and illustrates this possible link between PVB19 and CAP activation.

Conclusion
Renal diseases associated with PVB19 primary infection are diverse and infrequent. Although PVB19 primary infection is rare in adults, we suggest that PVB19 serology could be performed in patients with atypical presentations of TMA or glomerulonephritis, especially if they were preceded or associated with a viral syndrome, or occurred in an epidemic context. The diagnosis of PVB19 infection in these patients can spare an inadequate and potentially harmful immunosuppressive therapy.

Declarations
Ethics approval and consent to participate All procedures performed in this study were in accordance with the ethical standards of the institutional research committee (Institut National des Données de Santé) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent for the use of samples and clinical data was obtained from all individual participants included in the serum biocollection (DC-2012-1704, which comprises blood and urine samples as well as renal biopsies). The institutional review board of AP-HM approved this study, and stated that, according to the French legislation on the protection of personal data, no registration number was needed for this study.

Consent for publication
The 4 case reports mentioned in this manuscript were not published previously. Written informed consent was obtained from each patient for publication of their results.

Availability of data and material
All data used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
Pr Olivier Moranne is an Editorial Board Member of BMC Nephrology. None of the other authors have any competing interests to declare.

Funding
No specific funding was received for this work.

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