We present a 10-year-old girl who received allogeneic hematopoietic stem cell transplant (HSCT) for Philadelphia chromosome (Ph)-positive acute lymphoblastic leukaemia (ALL) and subsequently developed cytomegalovirus (CMV) encephalitis, successfully treated with CMV-hyperimmune globulin (Figure 1).
The girl was diagnosed with ALL one year prior, and as a result of the positivity for the Ph chromosome, she underwent HSCT in first complete remission. The virological screening performed during the pre-transplant workup by quantitative polymerase chain reaction (PCR) showed a CMV viral load of 4.0 x 103 copies mm-3. The patient received antiviral treatment with ganciclovir, obtaining a complete virus clearance in a few days. Immediately before the HSCT, ganciclovir was replaced with valganciclovir.
After total body irradiation (TBI)-based myeloablative conditioning, the girl received 4.8 x 108 kg-1 bone marrow total nuclear cells from the 10/10 HLA-matched, CMV-positive, unrelated donor. Graft-versus-host disease (GVHD) prophylaxis was performed with tacrolimus and mycophenolate mofetil. The patient received continuous antiviral prophylaxis with valganciclovir, except between days -1 and +20. Neutrophil and platelet engraftment were achieved at day +15 and day +22, respectively. The peripheral blood chimerism analysis at day +30 proved full donor chimerism, and the absence of BCR/ABL transcript in the bone marrow cells confirmed the complete molecular remission.
The patient could not start the maintenance treatment with tyrosine kinase inhibitors because of suboptimal graft function. She showed a marked lymphocyte depletion with a total lymphocyte count of 206 mm-3, CD3 4 mm-3, CD4 2 mm- 3, CD8 1 mm- 3 and CD19 4 mm- 3 at day +150. For this reason, and because the loads of CMV, Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6) and BKV remained low, the immunosuppressive regimen was discontinued.
At day +164, she developed a persistent headache and high blood CMV, EBV, HHV6 and BKV virus loads were detected. Valganciclovir was shifted to foscarnet. Magnetic resonance imaging (MRI) of the brain did not show central nervous system (CNS) involvement.
Despite the rapid viral blood clearance, the headache worsened, and severe fatigue developed. To rule out CNS disease relapse, at day +178, a lumbar puncture was performed. The cerebrospinal fluid (CSF) analysis revealed a lymphocytic pleocytosis (68 cells mm-3), slightly elevated protein level (49 mg dL-1) and normal glucose level (39 mg dL-1), without evidence of blast cells (CD19+CD58+CD10++) on flow cytometry. Tests for CSF toxoplasma, panfungal PCR and JC virus (JCV) were negative. The CSF specimen was stored at -80 C, pending some other diagnostic hypotheses, and the girl continued her outpatient antiviral treatment with valganciclovir.
At day +203, the patient presented with high fever and further worsening headache and fatigue. Lansky performance status decreased from 100% to 60%. A second MRI, serum C reactive protein (CRP) and all viral blood determinations were negative, while the total lymphocyte count increased to 2000 mm-3 with > 600 CD3 mm-3 and > 200 CD4 mm-3. At day +210, the girl was admitted because of fourth cranial nerve palsy with vertical diplopia and positive Bielschowsky test. A brain MRI showed multiple focal lesions with T1 hypointensity and diffusion hyperintensity without enhancement after contrasting medium administration. These findings were suggestive of an immune reconstitution inflammatory syndrome (IRIS). Quantitative PCR on CSF for CMV, EBV, all herpesviruses, JCV, toxoplasma and panfungal PCR was performed and showed a very high CMV load (7.3 x 105 copies mm-3), whereas the CMV PCR on blood was negative. A CMV PCR was also performed on the frozen CSF sample, collected at day +178. The viral load in this sample was also very high (6.8 x 105 copies mm-3). To detect why the clinical and radiological pictures were so different between the two episodes, despite the similar CSF viral loads in both specimens, CSF inflammatory cytokines concentrations were measured. IL-6 value was very high (96.07 pg mL) only in the CSF sample collected during the onset of neurological symptoms, suggesting a robust immune restoration and a likely CMV-induced IRIS.
The girl was started on a combined dual antiviral therapy with ganciclovir (10 mg kg-1 day-1), foscarnet (1800 mg kg-1 day-1) and weekly CMV-hyperimmune globulin (Cytotect CP, Biotest, Germany) infusions (200 U kg-1 dose-1). All the neurological symptoms began to improve right away. Unfortunately, the CSF viral load did not drop significantly after two, four and eight weeks of dual therapy. The possibility of a drug-resistant CMV strain was ruled out by the genotypic resistance test, which was aimed at detecting specific mutations in UL97 and UL54 genes.
At day +266, after two months of dual antiviral therapy, the girl developed severe renal impairment, neutropenia and thrombocytopenia. The CSF viral load was still very high, despite the complete clinical and radiological recovery. To avoid further renal and bone marrow toxicity, the dual antiviral therapy was suspended and Cytotect CP treatment alone was continued, at an increased frequency of 200 U kg-1 dose-1 for five consecutive days every week. At day +287, the CSF viral load dropped to 900 copies mm-3 and was undetectable at day +308. In the meantime, the renal impairment resolved, and there was a complete bone marrow recovery. On discharge, the girl was in excellent clinical condition, and she continued the CMV-hyperimmune globulin prophylaxis once a week for two months as an outpatient. Five years out of HSCT, the girl remains in perfect health and has not experienced any further CMV reactivations. All data regarding blood and CSF viral loads are displayed in Table 1.
During the Cytotect CP treatment, we measured the CMV-IgG antibody levels in the blood and CSF of our patient. The CNS antibody levels were significantly higher than those in blood in all the tests. For comparison, we conducted a retrospective analysis based on plasma and CSF samples stored in our biobank. From our records, we identified 30 CMV-IgG-positive paediatric patients affected by haematologic malignancies who had undergone standard treatment protocols and for whom both plasma and CSF samples were stored. None of these patients had CMV-related CNS involvement. All samples were obtained 3-5 months post-transplant. A minimum of three peripheral blood samples were collected for each patient. Except for the patient featured in this paper, there was only a single CSF sample for each of the patients. We divided selected patients into two comparable groups of 15 each. The patients in the first group had received prophylactic treatment with Cytotect CP (Cytotect group), while those in the second group had not (Control group). We performed serum and CSF CMV-IgG antibodies titre evaluation on the frozen samples. Comparing the titres of CMV-IgG antibody in blood and CSF, we found a statistically significant correlation in antibody concentration within each group, with an inverse correlation in the Cytotect group (P < 0.001 for the Cytotect group, P < 0.0001 for Control group). Comparing the titres of CMV-IgG antibody in blood and CSF across both groups, a positive correlation was found in blood antibody concentration, while CSF antibody concentration showed an inverse correlation (P < 0.0001 for both). Comparing the CMV-IgG antibodies blood/CSF ratio, we found statistically significant differences in antibody concentrations between the two groups, with inverse correlation in the Cytotect group (P < 0.001 for Cytotect group, P < 0.0001 for Control group). Similarly, comparing blood and CSF CMV-IgG titres in both groups, the positive correlation was found in blood antibodies concentration, while CSF antibodies titre showed an inverse correlation (P < 0.0001 for both). The results are shown in Figure 2.