Milena Furione

Preemptive Therapy of EBV-Related Lymphoproliferative Disease after Pediatric Haploidentical Stem Cell Transplantation

The treatment of Epstein‐Barr virus (EBV)‐related post‐transplant lymphoproliferative disease (PTLD) after hematopoietic stem cell transplantation (HSCT) is still unsatisfactory. We conducted a prospective trial to evaluate the impact of routine EBV surveillance and preemptive treatment with the anti‐CD20 monoclonal antibody rituximab on the development of PTLD in pediatric recipients of extensively T‐cell depleted HSCT from an HLA‐haploidentical relative. Twenty‐seven patients were included in the surveillance program, 12 developed EBV DNA positivity, with 8 of 12 presenting with sustained viral DNA levels requiring treatment with rituximab. Treatment was well tolerated, and induced clearance of EBV DNA in all patients. However, 4/8 patients showed a new increase in EBV load, coincident with the emergence of CD20−/CD19+ B cells in peripheral blood, accompanied by overt PTLD in 3 patients. The latter cleared PTLD after receiving donor EBV‐specific cytotoxic T‐lymphocytes (CTLs), and persist in remission at a median 30‐month follow‐up. EBV‐specific T‐cell frequency, undetectable at time of EBV DNA positivity, was restored by T‐cell therapy to levels comparable with controls. We conclude that preemptive therapy with rituximab is safe, but only partly effective in haplo‐HSCT recipients. Patients who progress to PTLD under rituximab treatment can be rescued permanently by infusion of EBV‐specific CTLs.

Postinfectious inflammatory disorders Subgroups based on prospective follow-up

Background: Acute disseminated encephalomyelitis (ADEM) refers to a monophasic acute multifocal inflammatory CNS disease. However, both relapsing and site-restricted variants, possibly associated with peripheral nervous system (PNS) involvement, are also observed, and a systematic classification is lacking. Objective: To describe a cohort of postinfectious ADEM patients, to propose a classification based on clinical and instrumental features, and to identify subgroups of patients with different prognostic factors. Methods: Inpatients of a Neurologic and Infectious Disease Clinic affected by postinfectious CNS syndrome consecutively admitted over 5 years were studied. Results: Of 75 patients enrolled, 60 fulfilled criteria for ADEM after follow-up lasting from 24 months to 7 years. Based on lesion distribution, patients were classified as encephalitis (20%), myelitis (23.3%), encephalomyelitis (13.3%), encephalomyeloradiculoneuritis (26.7%), and myeloradiculoneuritis (16.7%). Thirty patients (50%) had a favorable outcome. Fifteen patients (25%) showed a relapsing course. Poor outcome was related with older age at onset, female gender, elevated CSF proteins, and spinal cord and PNS involvement. All but two patients received high-dose steroids as first-line treatment, with a positive response in 39 (67%). Ten of 19 nonresponders (53%) benefited from high-dose IV immunoglobulin; 9 of 10 had PNS involvement. The data were not controlled. Conclusions: A high prevalence of “atypical variants” was found in this series, with site-restricted damage or additional peripheral nervous system (PNS) involvement. Prognosis and response to steroids were generally good, except for some patient subgroups. In patients with PNS involvement and steroid failure, a favorable effect of IV immunoglobulin was observed.

Diagnosis and Outcome of Preconceptional and Periconceptional Primary Human Cytomegalovirus Infections

Primary human cytomegalovirus (HCMV) infection occurring in pregnant women within 3 months before (preconceptional) or within 4 weeks after (periconceptional) the last menstrual period represents an as-yet-undefined risk to the fetus. One (9.1%) of 11 newborns born to 12 women with preconceptional infection was subclinically infected (1 aborted fetus was not examined for infection). Of 20 pregnancies in women with periconceptional infection, 7 were terminated before 12 weeks of gestation (aborted fetus was not examined), 1 was terminated at 23 weeks after prenatal diagnosis of congenital infection, and 12 continued to term. Of those 12, 3 resulted in newborns who were congenitally infected. Thus, in the periconceptional group, intrauterine transmission occurred in 4 (30.8%) of 13 pregnancies for which the virologic outcome was known. One newborn was symptomatic at birth, and disseminated HCMV infection was diagnosed in an aborted fetus. Periconceptional primary HCMV infection seems to bear a higher risk of unfavorable outcome than preconceptional infection, and counseling should be adjusted accordingly.

Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes of immunocompromised patients by the polymerase chain reaction

Human cytomegalovirus (HCMV) DNA amplification by the polymerase chain reaction (PCR) was utilized previously for successful monitoring of HCMV infections in immunocompromised patients. However, analysis of an extended series of clinical samples revealed the relatively frequent presence of PCR inhibitors. Hence, the need for availability of an internal control of the reaction allowing identification of false negative results. Similarly, an internal standard appeared necessary for quantification of viral DNA in clinical samples. For this purpose, we constructed a recombinant DNA molecule which could be amplified by the same set of primers used for HCMV DNA amplification. Coamplification of the recombinant DNA molecule and clinical samples proved to be a simple and reliable method for verifying sample competence for amplification. In addition, coamplification of serial known amounts of the same molecule, used as internal standard, and test sample, allowed quantification of viral DNA in polymorphonuclear leukocyte samples. Quantitative monitoring of HCMV infection and antiviral treatment may provide critical indications as to whether and when to initiate or discontinue antiviral treatment in immunocompromised patients with systemic HCMV infections.

Role of prenatal diagnosis and counseling in the management of 735 pregnancies complicated by primary human cytomegalovirus infection: A 20-year experience

BACKGROUND The burden of congenital human cytomegalovirus (HCMV) infection is well recognized. However, screening for maternal infection remains controversial in view of diagnostic challenges, counseling difficulties, and absence of medical treatment. OBJECTIVE To assess the role of prenatal diagnosis and counseling in the management of pregnancy complicated by primary HCMV infection. STUDY DESIGN Retrospective study aimed at investigating diagnostic features, options, and pregnancy outcome in 735 women with primary HCMV infection over a period of 20 years (1990-2009). RESULTS Overall, 25.6% women were found to be seronegative before the actual pregnancy. However, none were informed about HCMV infection and potential prevention strategies. Diagnosis of primary HCMV infection was achieved by seroconversion in 44.4% cases and by different combinations of virus-specific IgM, low IgG avidity, and DNAemia in 43.9% cases. Non-specific symptoms and/or haematological/biochemical alterations were recalled by 73.5% women. The onset of infection could be established, and counseling adjusted accordingly in >90% cases. The overall rate of vertical transmission was 37.1%, ranging from 5.6% for preconceptional infections to 64.1% for third trimester infections. Amniocentesis was chosen by 43.1% women, whereas pregnancy termination was requested by 15.6%. CONCLUSIONS Reference virology centers and ad hoc trained and experienced physicians are required for accurate diagnosis of primary infection in pregnancy and ensuing counseling. Prenatal diagnosis has a central role in the management of pregnancies complicated by primary HCMV infection. HCMV-seronegative women should receive adequate information.

Development of Human Cytomegalovirus—Specific T Cell Immunity during Primary Infection of Pregnant Women and Its Correlation with Virus Transmission to the Fetus

OBJECTIVE We sought to study the development of human cytomegalovirus (HCMV)-specific T cell-mediated immune responses during primary HCMV infection in pregnancy. METHODS The HCMV-specific lymphoproliferative response (LPR) and intracellular cytokine (interferon [IFN]- gamma and interleukin [IL]-2) production were investigated during the first year after primary infection in 49 pregnant women and 9 nonpregnant control subjects. An HCMV-specific CD4(+) and CD8(+) T cell LPR was detected by the 5,6-carboxyfluorescein diacetate succinimidyl ester dilution method, and a cell-division index was calculated. RESULTS The CD4(+) T cell LPR developed slightly earlier than the CD8(+) T cell LPR. However, CDI values for both T cell subpopulations were lower than those of seropositive control subjects in both pregnant and nonpregnant individuals. During the first month after infection, IFN- gamma -producing CD4(+) and CD8(+) T cells were consistently observed, whereas IL-2-producing T cells were very rarely detected in blood. A correlation between the development of HCMV-specific LPR and virus clearance from blood was observed. A significantly delayed development of the CD4(+) T cell LPR was observed in infected mothers who transmitted virus to the fetus, compared with those who did not. CONCLUSIONS The development of adaptive T cell immunity after primary HCMV infection appears to be a complex and slow process until a memory T cell response develops. The T cell immune response appears to influence vertical HCMV transmission.

Virologic and Immunologic Monitoring of Cytomegalovirus to Guide Preemptive Therapy in Solid‐Organ Transplantation

Control of human cytomegalovirus (HCMV) infection during the posttransplant period was investigated in 134 solid‐organ transplant recipients by monitoring in parallel virologic and immunologic parameters for at least 1 year of follow‐up. Virologic monitoring was achieved by determining HCMV DNAemia with real‐time PCR, using the threshold of 300 000 DNA copies/mL blood as a cutoff for starting preemptive therapy. Immunologic monitoring included measurement of HCMV‐specific CD4+ and CD8+ T cells by cytokine flow cytometry, using HCMV‐infected dendritic cells as a stimulus. HCMV infection was diagnosed in 110 (82%) and required treatment in 49 (36%) patients. At 12 months after transplantation ‘protective’ immunity (≥0.4 CD4+ and CD8+ HCMV‐specific T cells/μL blood) was achieved in 115/129 (89%) patients. During the entire study period, 122 patients reconstituting HCMV‐specific CD4+ and CD8+ T‐cell immunity at 60 days posttransplant onward were able to control HCMV infection, except for one patient who developed HCMV disease because of a rejection episode. Patients reconstituting HCMV‐specific CD8+ only did not control HCMV infection. In conclusion, the presence of both HCMV‐specific CD4+ and CD8+ T cells ≥ 0.4/μL blood appears to be protective against HCMV disease. This result does not apply to patients undergoing antirejection treatment, or reconstituting HCMV‐specific CD8+ T cells only.

Quantitation of human cytomegalovirus DNA in bone marrow transplant recipients

Summary. HCMV DNA was retrospectively quantitated in the early post‐transplant period in 36 paediatric bone marrow transplant (BMT) recipients prospectively monitored for human cytomegalovirus (HCMV) infection on the basis of antigenaemia and viraemia assays. Viral DNA was quantitated in peripheral blood leucocytes (PBL) by PCR using an internal control of amplification and a series of external standards. Densitometric analysis of hybridization results obtained on PCR products enabled construction of a standard curve from which DNA amounts of clinical samples, expressed in terms of genome equivalents (GE), were interpolated. Of the 36 BMT recipients, three had clinically symptomatic HCMV infection with mean peak levels of viral DNA > 5000 GE (antigenaemia and viraemia mean peak levels were 873 and 35, respectively), whereas 19 with HCMV reactivation were asymptomatic (five of them had abortive HCMV infection) showing mean peak DNA levels of 131 GE (and of 6‐8 and 13 for antigenaemia and viraemia, respectively) (P≤0.01). Single or multiple courses of pre‐emptive therapy with ganciclovir or foscarnet were given to 14/19 asymptomatic children in whom antigenaemia levels were > 2 or lower yet persisting. Overall, in the 14 asymptomatic treated patients the mean antigenaemia level was 9‐3 (range 1‐22), and the mean DNA level was 184‐6 (range 20‐710) GE. Antiviral drugs were also administered to the three symptomatic patients who, due to late diagnosis of HCMV infection, escaped preemptive therapy. Antiviral treatment caused marked decrease or disappearance of viral DNA, antigenaemia and viraemia in both symptomatic and asymptomatic patients. In conclusion, our study suggests that: (i) starting therapy in the presence of a mean antigenaemia level of 9′3 (range 1‐22) corresponding to a mean DNA level of 184′6 (range 20‐710) GE avoided occurrence of any major HCMV‐related clinical complication; (ii) clinical symptoms were associated with antigenaemia levels > 100 and DNA levels > 1000 GE; (iii) the effect of antiviral treatment could be more carefully monitored by quantitation of viral DNA.

The immunosuppression and potential for EBV reactivation of fludarabine combined with cyclophosphamide and dexamethasone in patients with lymphoproliferative disorders

Fludarabine is effective in chronic lymphocytic leukaemia (CLL) and low‐grade non‐Hodgkins lymphoma (NHL). A major side‐effect of this purine analogue is immunosuppression which may favour opportunistic infections. Additionally, impairment of immunosurveillance might promote Epstein‐Barr virus (EBV) reactivation and possibly favour transformation to high‐grade malignancy. The aim of this study was to evaluate the immunosuppression‐related effects of the fludarabine‐based combination Flucyd in advanced low‐grade NHL or CLL by serially monitoring T‐lymphocyte subsets, opportunistic infections, EBV‐reactivation, and histologic transformation. 24 patients with advanced NHL (n = 21) or CLL (n = 3) received fludarabine 25 mg/m2/d + cyclophosphamide 350 mg/m2/d + dexamethasone 20 mg/d in 3 d courses for a maximum of six courses. The overall response rate was 79% (eight CR, 11 PR, five failures); 11 patients relapsed or progressed between 3 and 19 months from response, and eight are in CR or PR at 3–27 months. The CD4+ lymphocyte counts decreased significantly during therapy from a median of 484/μl pre‐treatment (range 142–1865) to a median of 198/μl (71–367). In 19 responders monitored off therapy every 3 months until relapse/progression, CD4+ counts were persistently low with minimal recovery over time. During treatment, 16 infections occurred in 11/24 patients. No delayed opportunistic infections occurred in responders while off therapy. The circulating EBV DNA load serially measured in 19 patients by a quantitative PCR assay showed an increase in four patients during treatment. A lymph node biopsy performed in two of these was PCR positive for EBV DNA, whereas LMP1 and EBERs were negative. Six NHL patients evolved into high‐grade B‐cell NHL. In conclusion, fludarabine combined with cyclophosphamide and dexamethasone is an effective therapy for recurrent indolent lymphoma. This combination produces prolonged T‐lymphocytopenia and has the potential to reactivate a latent EBV infection. T‐cell dysfunction, however, is not associated with higher incidence of clinical opportunistic infections and does not adversely influence clinical outcome.

Prevention of Primary Cytomegalovirus Infection in Pregnancy.

Background Cytomegalovirus (CMV) is the leading infectious agent causing congenital sensorineural hearing loss and psychomotor retardation. CMV vaccine is currently unavailable and treatment options in pregnancy are limited. Susceptible pregnant women caring for children are at high risk for primary infection. CMV educational and hygienic measures have the potential to prevent primary maternal infection. Methods A mixed interventional and observational controlled study was conducted to investigate the effectiveness of hygiene information among pregnant women at risk for primary CMV infection for personal/occupational reasons. In the intervention arm, CMV-seronegative women, identified at the time of maternal serum screening for fetal aneuploidy at 11–12 weeks of gestation, were given hygiene information and prospectively tested for CMV until delivery. The comparison arm consisted of women enrolled at delivery who were neither tested for nor informed about CMV during pregnancy, and who had a serum sample stored at the screening for fetal aneuploidy. By design, groups were homogeneous for age, parity, education, and exposure to at least one risk factor. The primary outcome was CMV seroconversion. Acceptance of hygiene recommendations was a secondary objective and was measured by a self-report. Findings Four out of 331 (1.2%) women seroconverted in the intervention group compared to 24/315 (7.6%) in the comparison group (delta = 6.4%; 95% CI 3.2–9.6; P < 0.001). There were 3 newborns with congenital infection in the intervention group and 8 in the comparison group (1 with cerebral ultrasound abnormalities at birth). Ninety-three percent of women felt hygiene recommendations were worth suggesting to all pregnant women at risk for infection. Interpretation This controlled study provides evidence that an intervention based on the identification and hygiene counseling of CMV-seronegative pregnant women significantly prevents maternal infection. While waiting for CMV vaccine to become available, the intervention described may represent a responsible and acceptable primary prevention strategy to reduce congenital CMV.