Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays

The enzyme-linked immunospot (ELISPOT) assay has advanced into a useful and widely applicable tool for the evaluation of T-cell responses in both humans and animal models of diseases and/or vaccine candidates. Using synthetic peptides (either individually or as overlapping peptide mixtures) or whole antigens, total lymphocyte or isolated T-cell subset responses can be assessed either after short-term stimulation (standard ELISPOT) or after their expansion during a 10-day culture (cultured ELISPOT). Both assays detect different antigen-specific immune responses allowing the analysis of effector memory T cells and central memory T cells. This paper describes the principle of ELISPOT assays and discusses their application in the evaluation of immune correlates of clinical interest with a focus on the vaccine field

Sustained impairment of human cytomegalovirus (HCMV)-specific CD4+ and CD8+ T cell response is responsible for recurrent episodes of disseminated HCMV infection in a D+R- hand transplant recipient

Human cytomegalovirus (HCMV) infection is the major viral complication in solid organ transplant recipients. Seronegative recipents (R-) of organs from seropositive donors (D+) appear to be at higher risk of developing symptomatic HCMV infection. To what extent systemic life-threatening complications can be risked for non-life-saving transplant procedures? A case report describing successful treatment of repeated episodes of active HCMV infection in a D+R- hand recipient in the absence of HCMV-specific T-cell immunity is presented. In the attempt to save both the patient and the transplanted hand, a preemptive treatment strategy was adopted with the aim to boost the constitution of the virus-specific T-cell immune response and simultaneously avoid onset of disease. Careful monitoring of HCMV load in blood and HCMV-specific T-cell immunity guided administration of repeated courses of antiviral treatment and avoided emergence of HCMV-related symptoms. Following establishment of HCMV-specific CD4+ and CD8+ T-cell response, preemptive treatment was no longer required due to sustained HCMV disappearance from blood. The patient is now well, and his hand too. In conclusion, evaluation of virus-specific T-cell immunity is of crucial importance in D+R- transplant recipients and careful monitoring of HCMV-specific T cell mediated response should always parallel monitoring of HCMV load in transplant recipients

Onset of valganciclovir resistance in two infants with congenital cytomegalovirus infection

Ganciclovir and its prodrug valganciclovir are elective treatments for cCMV. Neonates with important symptoms undergo 6 months of therapy to ameliorate/prevent symptoms and late sequelae, but evidence of resistance is emerging. Over the last 5 years, we took care of 59 cCMV infants and experienced two cases of resistance among nine cCMV infants receiving long-term valganciclovir therapy. In the first case, valganciclovir therapy was prolonged beyond 6 months due to severity of symptoms, control of viral load, and absence of adverse events. Resistance was detected in the 8th month of therapy. In the second case, after a significant reduction following valganciclovir administration and no adverse events, CMV viral load suddenly increased in the 6th month of therapy due to resistance. Both events were associated with UL97 gene mutation. The cCMV infants, affected by severe symptoms, remained in a steady state during treatment, and their later neurological development was coherent with initial seriousness of diagnosis. Prolonged therapeutic exposure may therefore be a risk for resistance, suggesting that constant dosage/weight adjustments, monthly surveillance of viral load, and therapeutic drug monitoring could be proposed to monitor resistance onset and optimize the therapy regime. The risk–benefit ratio for long-term therapy, including the possibility of resistance onset, alongside SNHL and neurodevelopmental improvement, should also be evaluated

Monitoring for cytomegalovirus and Epstein-Barr virus infection in chronic lymphocytic leukemia patients receiving i.v. fludarabine-cyclophosphamide combination and alemtuzumab as consolidation therapy.

The combination of fludarabine and cyclophosphamide (FC) has become the standard of care in chronic lymphocytic leukemia (CLL) patients. Due to the well-recognized F-related immunosuppression,[1][1] a higher risk of opportunistic infections could be expected by adding another immunosuppressive agen

Hodgkin's disease as unusual presentation of post-transplant lymphoproliferative disorder after autologous hematopoietic cell transplantation for malignant glioma

BACKGROUND: Post-transplant lymphoproliferative disorder (PTLD) is a complication of solid organ and allogeneic hematopoietic stem cell transplantation (HSCT); following autologous HSCT only rare cases of PTLD have been reported. Here, a case of Hodgkin's disease (HD), as unusual presentation of PTLD after autologous HSCT for malignant glioma is described. CASE PRESENTATION: 60-years old man affected by cerebral anaplastic astrocytoma underwent subtotal neurosurgical excision and subsequent high-dose chemotherapy followed by autologous HSCT. During the post HSCT course, cranial irradiation and corticosteroids were administered as completion of therapeutic program. At day +105 after HSCT, the patient developed HD, nodular sclerosis type, with polymorphic HD-like skin infiltration. CONCLUSION: The clinical and pathological findings were consistent with the diagnosis of PTLD

Genetic divergence of influenza A NS1 gene in pandemic 2009 H1N1 isolates with respect to H1N1 and H3N2 isolates from previous seasonal epidemics

<p>Abstract</p> <p>Background</p> <p>The Influenza A pandemic sustained by a new H1N1 variant (H1N1v) started in Mexico and the USA at the end of April 2009 spreading worldwide in a few weeks. In this study we investigate the variability of the NS1 gene of the pandemic H1N1v strain with respect to previous seasonal strains circulating in humans and the potential selection of virus variants through isolation in cell culture.</p> <p>Methods</p> <p>During the period April 27^th2009-Jan 15^th2010, 1633 potential 2009 H1N1v cases have been screened at our center using the CDC detection and typing realtime RT-PCR assays. Virus isolation on MDCK cells was systematically performed in 1/10 positive cases. A subset of 51 H1N1v strains isolated in the period May-September 2009 was selected for NS1 gene sequencing. In addition, 15 H1N1 and 47 H3N2 virus isolates from three previous seasonal epidemics (2006-2009) were analyzed in parallel.</p> <p>Results</p> <p>A low variability in the NS1 amino acid (aa) sequence among H1N1v isolates was shown (aa identity 99.5%). A slightly higher NS1 variability was observed among H1N1 and H3N2 strains from previous epidemics (aa identity 98.6% and 98.9%, respectively). The H1N1v strains were closely related (aa identity 92.1%) to swine reference strain (A/swine/Oklahoma/042169/2008). In contrast, substantial divergence (aa identity 83.4%) with respect to human reference strain A/Brevig Mission/1/1918 and previous epidemic strains H1N1 and H3N2 (aa identity 78.9% and 77.6%, respectively) was shown. Specific sequence signatures of uncertain significance in the new virus variant were a C-terminus deletion and a T215P substitution.</p> <p>Conclusions</p> <p>The H1N1v NS1 gene was more conserved than that of previous epidemic strains. In addition, a closer genetic identity of H1N1v with the swine than the human reference strains was shown. Hot-spots were shown in the H1N1v NS1 aa sequence whose biologic relevance remains to be investigated.</p

Human parechovirus type 5 neurological infection in a neonate with a favourable outcome: A case report

The majority of parechovirus A type 5 (PeV-A5) infections have been reported in patients with gastrointestinal syndromes. In contrast, a sepsis-like illness associated with PeV-A5 infection has been reported only anecdotally. Herein, we report the first case in Italy of a PeV-A5 neurological infection presenting in a neonate with a sepsis-like syndrome. The patient, a healthy male infant born at 41 weeks of gestation, was highly distressed and inconsolable, and had been crying persistently, with poor breastfeeding, since the previous day. From day 2 to day 4, the newborn was feverish with mild irritability; breastfeeding was preserved and regularly supported. His clinical condition progressively improved, with defervescence on day 4. He was discharged after 7 days, and neurological examination results indicated only mild impairment in visual fixation and vertical eye tracking and mild axial hypotonia. The Italian PeV-A5 strain was phylogenetically related to three strains detected in Denmark in 2012, as well as to one detected in Australia and one in Greece in 2015, with an average nucleotide identity of 97.9% (range 95.9–100.0%). Enterovirus/PeV infection in the newborn should be ruled out in cases of infants with unexplained fever and/or a sepsis-like syndrome and/or meningoencephalitis. An aetiological diagnosis is essential to avoid the unnecessary administration of antibiotics and to plan long-term follow-up until schooling. Keywords: Neonatal infections, Human parechovirus type 5, CSF, Molecular characterization, CNS infection, Phylogenetic analysi

Viral shedding in children infected by pandemic A/H1N1/2009 influenza virus

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to investigate viral shedding in otherwise healthy children with pandemic A/H1N1/2009 influenza in order to define how long children with pandemic A/H1N1/2009 influenza shed the virus, and also plan adequate measures to control the spread of the disease within households.</p> <p>Findings</p> <p>In 74 otherwise healthy children with pandemic A/H1N1/2009 influenza, nasopharyngeal swabs were taken for virus detection upon hospital admission and every two days until negative. The nasopharyngeal swabs of all of the children were positive for pandemic A/H1N1/2009 influenza virus in the first three days after the onset of infection, and only 21.6% and 13.5% remained positive after respectively 11 and 15 days. No child was positive after more than 15 days. Viral load also decreased over time, and was not associated with patient age or the risk of pneumonia. Those who shed the virus for ≥ 9 days were not at any increased risk of suffering from more severe disease in comparison with those who shed the virus for a shorter time, but their households experienced a significantly higher number of influenza-like illness during the two weeks after the onset of the initial disease (72.3% <it>vs </it>41.4%; p < 0.05).</p> <p>Conclusions</p> <p>Regardless of their age, healthy children can shed pandemic A/H1N1/2009 influenza virus for up to two weeks after illness onset, and the households of the children who shed the virus for ≥ 9 days suffered a higher number of influenza-like illness in the two weeks following the onset of the first disease. This could suggest that when a completely unknown influenza virus is circulating, isolation period of infected children has to be longer than the 7 days recommended for the infections due to seasonal influenza viruses.</p

Antibody response of healthy children to pandemic A/H1N1/2009 influenza virus

<p>Abstract</p> <p>Background</p> <p>Little is known about the proportion of pediatric pandemic A/H1N1/2009 influenza cases who showed seroconversion, the magnitude of this seroconversion, or the factors that can affect the antibody level evoked by the pandemic A/H1N1/2009 influenza. Aims of this study were to analyse antibody responses and the factors associated with high antibody titres in a cohort of children with naturally acquired A/H1N1/2009 influenza infection confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR).</p> <p>Results</p> <p>Demographic, clinical and virologic data were collected from 69 otherwise healthy children with pandemic A/H1N1/2009 influenza (27 females, mean age ± SD: 5.01 ± 4.55 years). Their antibody levels against pandemic A/H1N1/2009 and seasonal A/H1N1 influenza viruses were evaluated by measuring hemagglutination-inhibiting antibodies using standard assays. Sixty-four patients (92.8%) with pandemic A/H1N1/2009 influenza had A/H1N1/2009 antibody levels of ≥40, whereas only 28/69 (40.6%) were seroprotected against seasonal A/H1N1 influenza virus. Those who were seroprotected against seasonal A/H1N1 virus were significantly older, significantly more often hospitalised, had a diagnosis of pneumonia significantly more frequently, and were significantly more often treated with oseltamivir than those who were not seroprotected (<it>p </it>< 0.05). The children with the most severe disease (assessed on the basis of a need for hospitalisation and a diagnosis of pneumonia) had the highest antibody response against pandemic A/H1N1/2009 influenza virus.</p> <p>Conclusions</p> <p>Otherwise healthy children seem to show seroprotective antibody titres after natural infection with pandemic A/H1N1/2009 influenza virus. The strength of the immune response seems to be related to the severity of the disease, but not to previous seasonal A/H1N1 influenza immunity.</p