Vanina Rognoni

Correlation of Rhinovirus Load in the Respiratory Tract and Clinical Symptoms in Hospitalized Immunocompetent and Immunocompromised Patients

While human rhinoviruses (HRVs) are well accepted as a major cause of common cold syndromes (rhinitis), their role in the etiology of lower respiratory tract infections is still controversial, and their detection in asymptomatic patients is relatively common. The HRV pathogenic role in four groups of hospitalized patients (pediatric immunocompetent and immunocompromised patients, and adult immunocompetent and immunocompromised patients) was investigated by quantifying HRV load in nasopharyngeal aspirates or bronchoalveolar lavage samples by real‐time reverse transcription PCR (RT‐PCR). Real‐time RT‐PCR was performed in duplicate on all respiratory samples resulting positive by qualitative RT‐PCR. In addition, molecular typing allowed detection of all known HRV species (A, B, and C). In immunocompetent pediatric patients HRVs were mostly associated with lower respiratory tract infections (in the absence of other viral agents) and wheezing, when viral load was ≥106 RNA copies/ml. In young immunocompromised patients (stem cell transplantation recipients), an inverse correlation between HRV persistence over time and time at which the infection occurred after transplantation was observed, whereas in adult immunocompromised patients (lung transplant recipients) HRVs could be detected at a medium–low level (<105 RNA copies/ml) in bronchoalveolar lavage samples taken routinely from asymptomatic patients. In conclusion, when detected at high viral load, HRVs may cause severe upper and lower respiratory tract infections, whereas when detected at a medium–low viral load, an event more frequent in immunocompromised subjects, they may represent only bystander viruses. J. Med. Virol. 81:1498–1507, 2009.

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.

Monitoring of Human Cytomegalovirus and Virus- Specific T-Cell Response in Young Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation

In allogeneic hematopoietic stem-cell transplantation (HSCT) recipients, outcome of human cytomegalovirus (HCMV) infection results from balance between viral load/replication and pathogen-specific T-cell response. Using a cut-off of 30,000 HCMV DNA copies/ml blood for pre-emptive therapy and cut-offs of 1 and 3 virus-specific CD4+ and CD8+ T cells/µl blood for T-cell protection, we conducted in 131 young patients a prospective 3-year study aimed at verifying whether achievement of such immunological cut-offs protects from HCMV disease. In the first three months after transplantation, 55/89 (62%) HCMV-seropositive patients had infection and 36/55 (65%) were treated pre-emptively, whereas only 7/42 (17%) HCMV-seronegative patients developed infection and 3/7 (43%) were treated. After 12 months, 76 HCMV-seropositive and 9 HCMV-seronegative patients (cumulative incidence: 90% and 21%, respectively) displayed protective HCMV-specific immunity. Eighty of these 85 (95%) patients showed spontaneous control of HCMV infection without additional treatment. Five patients after reaching protective T-cell levels needed pre-emptive therapy, because they developed graft-versus-host disease (GvHD). HSCT recipients reconstituting protective levels of HCMV-specific T-cells in the absence of GvHD are no longer at risk for HCMV disease, at least within 3 years after transplantation. The decision to treat HCMV infection in young HSCT recipients may be taken by combining virological and immunological findings.

Kinetics of human cytomegalovirus (HCMV) DNAemia in transplanted patients expressed in international units as determined with the Abbott RealTime CMV assay and an in-house assay.

BACKGROUND Consensus human cytomegalovirus (HCMV) DNA cut-off values for preemptive therapy in transplant recipients have not yet been defined, mainly due to the lack of real-time PCR standardization. OBJECTIVES (i) To compare the kinetics of HCMV DNA in transplanted patients using an in-house real-time PCR assay (Pavia assay) and the new Abbott RealTime CMV assay; (ii) to verify assay concordance in the identification of patients eligible for preemptive treatment and (iii) standardize results with international units (IUs) using the WHO International HCMV DNA Standard as a reference. STUDY DESIGN The kinetics of HCMV disseminated infection was retrospectively evaluated in 513 stored whole blood samples from 37 transplanted patients enrolled in randomized prospective studies designed for the clinical validation of HCMV DNA cut-off values. Conversion factors of HCMV DNA copy number to WHO international units (IUs) were determined. RESULTS Among the 513 samples, 352 (68.6%) were concordant positive, 42 (8.1%) concordant negative and 119 (23.1%) discordant. All discordant samples resulted positive by the Abbott RealTime CMV assay and negative by the Pavia assay, showing higher sensitivity for the Abbott RealTime CMV assay. A significant correlation was observed between concordant positive samples (r=0.89). HCMV DNAemia determined by each assay showed overlapping kinetics. Expression of results as IU/ml provided the best results in preemptive treatment simulation. CONCLUSION HCMV DNAemia cut-off values determined using our in-house assay and expressed as IU/ml appear valid for use in commercial assays as well as other potential in-house assays.

Post-transplant lymphoproliferative disorders and Epstein-Barr virus DNAemia in a cohort of lung transplant recipients

BackgroundPost-transplant lymphoproliferative disorders (PTLD) are serious complications in lung transplant recipients. No consensus on EBV DNAemia levels predictive of PTLD has been reached. In addition, in many instances EBV DNAemia is determined in patients with suggestive symptoms only.MethodsThe characteristics of five patients with PTLD as well as the prevalence of EBV DNAmia in a cohort of 137 consecutive patients receiving lung transplantation are described.ResultsTwenty-six out of 137 patients (18.9%) were excluded from the analysis because lost at follow-up or dead from PTLD-independent reasons within three months of transplantation. EBV DNA in peripheral blood mononuclear cells (PBMC) was determined in 83/111 patients (74.8%) because of potential PTLD-related symptoms, while 28 patients (25.2%) showed no symptoms and were not examined. EBV DNAemia was positive in 53/83 patients (63.8%), and negative in 30/83 patients (36.2%). PTLD was diagnosed in five (4.5%) patients at a median time of 270 (range 120-870) days following transplantation. All five PTLD (three large B-cell lymphomas, one Hodgkin lymphoma and one possible pre-neoplastic lesion) were potentially associated with EBV infection. However, only 3/5 patients with PTLD had detectable EBV DNAemia: < 1,000 copies EBV DNA/1 × 105 PBMC in one patient and > 1,000 copies EBV DNA/1 × 105 PBMC in two patients.ConclusionA systematic multidisciplinary (clinical, radiologic, virologic and histologic) approach is mandatory for the diagnosis and management of PTLD in lung transplant recipients, while monitoring of symptomatic patients only may provide an incomplete or late picture of the clinical problem. In addition, staining for EBV antigens and quantification of EBV DNA in biopsy specimens should always be performed to understand the role of EBV infection in the pathogenesis of PTLD.

Cytomegalovirus DNAemia in pregnant women

BACKGROUND Cytomegalovirus (CMV) transmission from mother to fetus occurs at a much greater rate following primary rather than reactivated infections and CMV dissemination in the mother is considered a key step in the pathogenesis of fetal infection. However, knowledge of CMV DNAemia in CMV-seropositive pregnant women is very limited. OBJECTIVE Major objective of this study was to assess the prevalence and diagnostic value of CMV DNAemia in a large population of seropositive pregnant women. STUDY DESIGN Serologic and DNAemia results obtained from 2211 blood samples of 1371 consecutive pregnant women referred to our Institution for suspected CMV infection in the period 2001-2010 were reviewed. RESULTS DNAemia was detected in 452/597 (75.7%) women with serologic evidence of primary CMV infection and in 4/774 (0.5%) women without evidence of primary infection. CONCLUSION In pregnant women, CMV DNAemia is detected primarily during primary infection. CMV DNAemia determination may be helpful in the diagnosis of primary infection.

Maternal, fetal, and neonatal parameters for prognosis and counseling of HCMV congenital infection

To investigate retrospectively the prognostic significance of maternal, fetal, and neonatal parameters and clinical outcome in 150 HCMV congenital infections during the period 1995–2009. HCMV fetal infection was investigated in amniotic fluid and fetal blood samples. HCMV congenital infection was confirmed in newborn urine and blood samples. Symptomatic infection was defined in HCMV‐infected fetuses and in infected newborns on the basis of physical and instrumental findings. Follow‐up at 3, 6, 12 months, and then annually up to school age, included clinical evaluation, funduscopic, audiologic, neurologic, and cognitive assessment. Overall, 122/150 (81.3%) newborns were asymptomatic and 28/150 (18.7%) were symptomatic at birth. The best prognostic maternal parameter of symptomatic infection at birth was gestational age at infection (P = 0.037). The best fetal virological markers were HCMV DNA levels in amniotic fluid (P < 0.001), antigenaemia levels (P = 0.007), HCMV DNA levels in blood (P = 0.004), and HCMV‐specific IgM index values (P = 0.002). The only significant neonatal parameter was HCMV DNA level in blood [P = 0.006; OR, 3.62 (95% CI, 1.46–8.97)]. Symptoms at birth correlated significantly with long‐term sequelae (P = 0.021). A trend towards a risk of sequelae in early (n = 15/58 examined) versus late (n = 6/57 examined) maternal infection was documented. The risk of symptomatic congenital infection at birth increased linearly with the number of significant maternal, fetal, and neonatal parameters. J. Med. Virol. 86:2163–2170, 2014.

Human cytomegalovirus-specific CD4+ and CD8+ T-cell response determination: Comparison of short-term (24 h) assays vs long-term (7-day) infected dendritic cell assay in the immunocompetent and the immunocompromised host

Human cytomegalovirus (HCMV)-specific CD4(+) and CD8(+) T-cells were measured in the immunocompetent host as well as in 13 solid-organ transplant recipients (SOTR), and 12 young hematopoietic stem cell transplant recipients (HSCTR) by using a long-term (7-day) assay based on PBMC stimulation by HCMV-infected dendritic cells (iDC), and two short-term (24h) assays, one for CD4(+) stimulation by infected cell lysate (iCL), and the other for CD8(+) stimulation by a pool of 34 epitopic peptides (pep-pool). In the immunocompetent, the number of T-cells activated by either iCL or the pep-pool was significantly reduced with respect to iDC. In both SOTR and HSCTR, the number of T-cells activated by iDC was comparable to that activated by iCL or the pep-pool. A significant correlation between iDC-activated T-cells and T-cells activated by either iCL or the pep-pool was observed. In conclusion, whenever a rapid result is needed, short-term assays may efficiently replace the iDC assay.

Differential outcome of neurological HCMV infection in two hematopoietic stem cell transplant recipients

BackgroundHuman cytomegalovirus (HCMV) infection of the central nervous system (CNS) is a rare but life threatening condition which may follow hematopoietic stem cell transplantation. Diagnosis, monitoring and treatment approaches rely on anecdotal reports.Case presentationsThe different outcomes of HCMV CNS disease in an adult and a pediatric T-cell depleted hematopoietic stem cell transplant (HSCT) recipient are reported. In the first case, HCMV encephalitis emerged in the context of simultaneous impairment of the T- and B-cell immunity. Antiviral treatment only reduced viral load in peripheral blood and the patient died. In the second case, an HCMV radiculopathy was observed and antiviral treatment was adjusted on the basis of intrathecal drug level. In addition, donor HCMV-specific cytotoxic T lymphocytes (CTLs) were infused. Viral load in the CNS decreased and the patient recovered from the acute event. In neither case were drug-resistant HCMV variants observed in blood or CNS samples.ConclusionsT-cell depleted HSCT appears a predisposing condition for CNS HCMV infection since never observed in other HSCT recipients at our center in the last 15 years. Intensive diagnostic approaches and timely aggressive combination treatments might improve clinical outcome in these patients.