Multidrug-Resistant Cytomegalovirus Infection in a Patient with Granulomatosis with Polyangiitis during Immunosuppressive Treatment

Introduction

Cytomegalovirus (CMV) is the most frequently opportunistic viral pathogen associated with mortality and morbidity in autoimmune disorders. However, the incidence and the clinical implications of CMV disease in this population has been thus far poorly investigated [1,2]. In a study involving 151 patients with rheumatic diseases including polyangiitis and treated with high-dose steroids associated with cyclophosphamide (CPA), fatal outcomes from CMV disease were reported in 29% of cases, highlighting the potential clinical impact of CMV infection in these patients [3].

The following report describes the case of a patient affected by granulomatosis with polyangiitis (GPA), a small-vessel vasculitis, treated with high-dose steroid and CPA for underlying disease, who subsequently developed a persistent and multidrug-resistant (MDR) CMV infection.

Case Report

In February 2014, a 54-year-old woman was admitted to the Infectious Diseases Unit at the Hospital of Ferrara, after a 2-week history of coughing, diarrhoea and fever unresponsive to antibiotic treatments. A GPA with renal, cerebral and pulmonary involvement had been diagnosed 1 year before and treated with intravenous (iv) CPA 100 mg/day and pulse glucocorticoids (methylprednisolone at 500 mg daily for 6 days followed by prednisone 60 mg daily for 2 weeks, 40 mg daily for 2 weeks, 30 mg daily for 2 weeks and 20 mg daily for 5 weeks) and then by CPA and oral prednisone (25 mg daily), which was still ongoing at time of hospital admission. Laboratory findings revealed pancytopenia and acute renal failure (creatinine 4.5 mg/dl). The CD4/CD8 ratio was not feasible due to lymphopenia (that is, absolute lymphocyte count <500 cells/ml; CPA cumulative dose 48.75 g). Blood, urine and stool cultures, QuantiFERON-TB test, Vidal-Wright and HIV serology were negative. Whole blood, urine and stool samples tested positive for CMV DNA with levels equal to 1,943,810 copies/ml, 1,600 copies/ml and 60 copies/μg DNA, respectively, revealing a widespread systemic CMV infection in the already CMV-seropositive patient.

Ganciclovir (GCV) at a dosage of 2.5 mg/kg twice daily, was promptly administered according to the renal filtrate, while CPA and steroid treatments were stopped. CMV DNAemia levels rapidly decreased and the patient showed a progressive improvement of gastrointestinal and renal function (creatinine 2.3 mg/dl). The patient was discharged after 21 days, due to the complete remission of symptoms, starting valganciclovir treatment (900 mg daily); CMV DNAemia was monitored at least approximately once a month. During the following 75 days, the patient did not present a clearance of viral load (median CMV DNAemia level: 1,666 copies/ml, range: 1,222–9,017), in absence of CMV-related symptoms. 1 month later, prednisone at 12.5 mg daily was reintroduced to avoid a possible reactivation of the autoimmune disease. In the meantime, the genotypic resistance testing was performed by Sanger sequencing of PCR-amplified UL97 and UL54 gene segments [4,5] and no drug-resistant (DR) CMV strain was found. During the next 3 months, a combined virological and immunological monitoring of CMV infection was performed, as previously described [6,7], by quantitative real-time PCR assay (CMV ELITe MGB™ kit, ELITech Group, Turin, Italy) and EliSpot assay (EliSpot Interferon-γ Basis kit; GenID GmbH, Strasburg, France), respectively. The immunological test enumerates interferon-gamma-secreting CMV-specific T-cells (both CD4⁺ and CD8⁺ cells), upon in vitro stimulation with viral antigens [7]. A persistent viral replication (median CMV DNA level: 3,992 copies/ml, range: 1,829–9,866) with weak CMV-specific CD4⁺ and CD8⁺ T-cell responses (13 spot-forming cells/200,000 peripheral blood mononuclear cells) was observed, in absence of CMV-related symptoms. During the following month, the patient was hospitalized due to onset of hyperpyrexia and drowsiness. A viral encephalitis was suspected by neurological assessment and then confirmed with magnetic resonance imaging (diffuse hyperintense areas, T2-weighted, in periventricular white matter semioval centre and juxta-cortical). Lumbar puncture was not performed due to severe thrombocytopenia. However, an increase of CMV DNAemia (79,800 copies/ml) was observed leading to treating the patient with iv foscarnet at 45 mg/kg every 8 h for 13 days and subsequently reduced to 50 mg/kg every 12 h for 3 days due to a worsening of renal function. A further CMV genotypic resistance testing disclosed two mutations: A594V (UL97 gene) and P522S (UL54 gene) conferring resistance to GCV and both GCV and cidofovir (CDV), respectively. Despite a decrease of viral load (6,112 copies/ml), the patient died due to multi-organ failure, secondary to acute renal failure after 16 days from hospitalization. During the follow-up period, no lesion referable to underlying disease reactivation was disclosed by rheumatological assessments and instrumental examinations (thoracoabdominal and brain computed tomography scans).

Of note, genotypic resistance testing was retrospectively performed on two different blood samples collected at the time of first hospitalization and 35 days before the detection of the two mutations. The results showed mutant CMV strain (A594V, UL97 gene) only in the second sample. Immunological and virological data, including CMV drug resistance mutations, as well as antiviral and immunosuppressive therapies are shown in Figure 1.

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Figure 1

Temporal evolution of CMV load levels, CMV-specific T-cell immune responses and onset of viral mutations in relation to the administration of antiviral and immunosuppressive treatments

Discussion

The patient described in the case study developed an MDR CMV infection during immunosuppressive therapy for GPA. As reported by other authors, CMV infection is often overlooked in the rheumatic disease field despite the severe immunosuppression due to treatment regimens aimed at controlling the underlying disease [3]. Of note, CMV infection was suspected and confirmed only 2 weeks after the onset of symptoms, when bacterial infections were excluded. The clinical management of CMV infection has been established at the centre level, due to the lack of guidelines on this topic in patients with autoimmune disorders [2,3,8,9]. Specifically, GCV was promptly started and the immunosuppressive treatment was stopped. The efficacy of the applied therapeutic approach was confirmed by the rapid decrease of CMV DNAemia along with a complete disappearance of clinical symptoms. However, during the following 4 months, despite the patient remaining asymptomatic, the virological monitoring showed persistent CMV DNAemia levels under continuous antiviral treatment. As suggested by literature data [10], a genotypic resistance testing was performed and no CMV drug resistance mutation was identified. This condition was described by other authors as clinical resistance [11]. It can be hypothesized that the lack of CMV-specific T-cell responses could have contributed to the failure in achieving spontaneous viral clearance. Since the patient's clinical condition was stable and, considering the toxicity of the available alternative CMV treatments, the antiviral therapy was not switched. During the subsequent 4 months, despite observing a CMV-specific immune reconstitution, persistent low CMV DNAemia levels continued to be detected and the occurrence of CMV DR mutations was found. The latter event generally occurs after prolonged drug exposure (weeks to months) with incomplete viral suppression [12]. In particular, two viral mutations conferring MDR were detected: A594V (UL97 gene) and subsequently P522S (UL54 gene) that confer the increases equal to 8.3- and 3.1-fold in GCV resistance, respectively [5,13]. In addition, for P522S mutation, a 3.6-fold increase in CDV 50% inhibitory concentration was determined [13]. As suggested by other authors, P522S mutation may occur without specific drug exposure to CDV and usually tends to evolve after the A594V mutation in the UL97 gene. The resistance to GCV is mostly associated with mutations in the UL97 gene, while the GCV resistant UL54 mutations alone are rare, probably due to a reduced fitness of CMV strains with mutation in the UL54 gene [14]. It can be speculated that, despite the development of the GCV resistant A594V mutation, the reconstitution of the CMV-specific cellular immune responses, albeit weak, could have been able to maintain low CMV DNAemia levels. As reported in the post-transplant setting, the CMV-specific cell-mediated immunity is an essential host factor in the control of CMV infection [15]. Nevertheless, in presence of the dual UL97/ UL54 mutations that commonly confer high degrees of GCV resistance (>30-fold) [16], the immune response against CMV alone may not have been sufficient to control the viral replication. Although the adjustment of antiviral treatment to MDR against GCV and CDV led to a decrease of viral load, the patient died due to multi-organ failure presumably secondary to acute renal failure, though autopsy was not performed. In the described case, it is very difficult to distinguish the contribution of the direct effects of CMV infection and the toxicity due to the anti-CMV therapies on the patient's deteriorating clinical course. Moreover, even if the reactivation of the underlying disease had been repeatedly ruled out throughout the clinical course of CMV disease, the induction treatment of GPA with high doses of CPA, had irreversibly led to a damage of the medullary haematopoiesis, resulting in a severe pancytopenia and a late CMV-specific immune reconstitution that was unable to clear the infection. These findings affirm the importance of severe CMV infections in patients with autoimmune disorders, highlighting that virological surveillance of CMV infection by CMV DNAemia quantification should be performed in patients with immune impairment, in particular in cases of fever of unknown origin. Moreover, assessment of CMV-specific immune responses and CMV drug resistance testing can be useful tools to guide the therapeutic decision-making on the basis of the clinical scenario and/or in case of increasing or persistent viral load during antiviral therapy.

In the near future, the treatment of CMV infection with novel antiviral drugs such as letermovir and maribavir, in combination with current anti-CMV drugs or with immunomodulatory agents (for example, leflunomide) showing anti-CMV properties, could constitute an effective alternative therapeutic strategy to treat MDR CMV infection and potentially reduce the emergence of MDR CMV strains.

In conclusion, the successfully therapeutic management of MDR CMV infection remains a challenge that requires further investigation.