Jayant S. Kalpoe

Validation of Clinical Application of Cytomegalovirus Plasma DNA Load Measurement and Definition of Treatment Criteria by Analysis of Correlation to Antigen Detection

ABSTRACT Successful preemptive cytomegalovirus (CMV) therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections. The pp65 antigenemia assay has been used for this purpose with considerable success. Quantification of CMV DNA is currently regarded to be an alternative diagnostic approach. The precise relationship between these two methods has still to be defined, but is essential to compare diagnostic results. This study compared the results of both assays with a large series of transplant recipients in different categories. An internally controlled quantitative real-time CMV DNA PCR was used to test 409 plasma samples from solid organ transplant (SOT) and stem cell transplant (SCT) patients. Levels of CMV DNA in plasma correlated well with classified outcomes of the pp65 antigenemia test. Despite this correlation, the quantitative CMV PCR values in a class of antigen test results were within a wide range, and the definition of an optimal cutoff value for initiating treatment required further analysis by a receiver-operating characteristic curve analysis. This is essential for reactivating infections in particular. For the SCT patients the optimal cutoff value of CMV DNA load defining relevant viral reactivation (in this assay, 10,000 copies/ml) was slightly higher than that for the SOT patients (6,300 copies/ml). Based on a comparison with the established pp65 antigenemia assay, quantification of CMV DNA in plasma appeared to be capable of guiding the clinical management of transplant recipients. This approach may have important advantages, which include a superior reproducibility and sensitivity, allowing the inclusion of kinetic criteria in clinical guidelines.

Management of Epstein-Barr Virus (EBV) Reactivation after Allogeneic Stem Cell Transplantation by Simultaneous Analysis of EBV DNA Load and EBV-Specific T Cell Reconstitution

BACKGROUND Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation and may progress to life-threatening lymphoproliferative disease (EBV-LPD) in the absence of adequate EBV-specific T cell immunity. Quantification of EBV DNA load in asymptomatic individuals who are at risk is a useful (although not entirely predictive) indicator of progression to EBV-LPD and guide for preemptive treatment with CD20 antibodies. METHODS With the aim of improving the identification of patients at risk, we retrospectively analyzed, within a cohort of 25 consecutive allogeneic stem cell transplant recipients at risk for EBV-LPD, the pattern of T cell reconstitution during EBV reactivation in all preemptively treated patients (8 patients). RESULTS In 6 of 8 cases, a significant T cell reconstitution (i.e., a CD3+ T cell count of >300 cells/microL) was documented during EBV reactivation, which included an expansion of EBV-specific memory T cells, as shown by human leukocyte antigen class I tetramer analysis. Additional evidence for the antiviral potential of this T cell reconstitution was obtained prospectively from a cohort of 14 consecutive allogeneic stem cell transplant recipients at risk for EBV-LPD. EBV reactivation occurred in 3 patients. Preemptive treatment was successfully withheld for 2 of these patients in light of concurrent (EBV-specific) T cell recovery. CONCLUSION We conclude that analysis of the level of (EBV-specific) T cell reconstitution during EBV reactivation is an important second parameter, in addition to quantification of EBV DNA load, that will be instrumental in a more accurate definition of patients at risk for EBV-LPD who, given their immunoincompetence, will be most certainly dependent on preemptive interventions.

Oral valganciclovir as pre-emptive therapy has similar efficacy on cytomegalovirus DNA load reduction as intravenous ganciclovir in allogeneic stem cell transplantation recipients.

The efficacy and safety of oral valganciclovir was compared to ganciclovir i.v. in pre-emptive treatment of cytomegalovirus (CMV) in T-cell-depleted allogeneic stem cell transplant (allo-SCT) recipients. A therapeutic guideline was developed to allow the safe application of valganciclovir in allo-SCT recipients requiring CMV therapy. In total, 107 consecutive transplant recipients were evaluated. Cytomegalovirus DNA load in plasma was monitored longitudinally; details on antiviral therapy and treatment responses were analyzed retrospectively. Fifty-seven CMV treatment episodes were recorded in 34 patients: 20 with valganciclovir (900 mg twice-daily) and 37 with ganciclovir (5 mg/kg twice-daily). Median CMV DNA load reduction was 0.079 and 0.069 log10 copies/ml/day in the ganciclovir and valganciclovir group, respectively. Good response on CMV DNA load (reduction below 3.0 log10 copies/ml) was observed in 75.7% of ganciclovir and 80.0% of valganciclovir treatment episodes. Severe adverse effects were not observed and CMV-related disease did not occur. However, the percentage of patients receiving erythrocyte transfusion was higher in the group of patients receiving ganciclovir as compared to valganciclovir (41 versus 20%, P=0.116). In conclusion, pre-emptive treatment with valganciclovir and ganciclovir, led to similar reduction of CMV DNA load. Oral valganciclovir is an attractive and safe alternative for pre-emptive CMV treatment in T-cell-depleted allo-SCT recipients.

Assessment of disseminated adenovirus infections using quantitative plasma PCR in adult allogeneic stem cell transplant recipients receiving reduced intensity or myeloablative conditioning.

Objectives:  Disseminated adenovirus (AdV) infections following allogeneic stem cell transplantation (allo‐SCT) are increasingly recognised, particularly in children. This study evaluated the clinical relevance of disseminated AdV infections in adult allo‐SCT recipients, after different conditioning regimens.

Combined CD8+ and CD4+ adenovirus hexon-specific T cells associated with viral clearance after stem cell transplantation as treatment for adenovirus infection

Background Human adenovirus can cause morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation. Reconstitution of adenovirus-specific CD4+ T cells has been reported to be associated with sustained protection from adenovirus disease, but epitope specificity of these responses has not been characterized. Since mainly CD4+ T cells and no CD8+ T cells specific for adenovirus have been detected after allogeneic stem cell transplantation, the relative contribution of adenovirus-specific CD4+ and CD8+ T cells in protection from adenovirus disease remains to be elucidated. Design and Methods The presence of human adenovirus hexon-specific T cells was investigated in peripheral blood of pediatric and adult allogeneic stem cell transplant recipients, who showed spontaneous resolution of disseminated adenovirus infection. Subsequently, a clinical grade method was developed for rapid generation of adenovirus-specific T-cell lines for adoptive immunotherapy. Results Clearance of human adenovirus viremia coincided with emergence of a coordinated CD8+ and CD4+ T-cell response against adenovirus hexon epitopes in patients after allogeneic stem cell transplantation. Activation of adenovirus hexon-specific CD8+ and CD4+ T cells with a hexon protein-spanning peptide pool followed by interferon-γ-based isolation allowed rapid expansion of highly specific T-cell lines from healthy adults, including donors with very low frequencies of adenovirus hexon-specific T cells. Adenovirus-specific T-cell lines recognized multiple MHC class I and II restricted epitopes, including known and novel epitopes, and efficiently lysed human adenovirus-infected target cells. Conclusions This study provides a rationale and strategy for the adoptive transfer of donor-derived human adenovirus hexon-specific CD8+ and CD4+ T cells for the treatment of disseminated adenovirus infection after allogeneic stem cell transplantation.

Clinical relevance of quantitative varicella-zoster virus (VZV) DNA detection in plasma after stem cell transplantation

Detection of Varicella-Zoster virus (VZV) DNA in plasma can facilitate the early recognition of complicated VZV-infection in immunocompromised hosts. The correlation of VZV-DNA in plasma with clinical presentations of VZV-infection and subsequent aciclovir treatment in allogeneic stem cell transplant (allo-SCT) recipients was studied. In 81 consecutive VZV-IgG positive allo-SCT recipients, VZV-DNA was measured at regular time points (1, 2 and 4 months) following allo-SCT and patient records were screened for VZV-related symptoms and aciclovir treatment. Subsequently, possible VZV-cases were studied in detail for the course of VZV-DNA and treatment effects. During the initial screening, VZV-DNA was detectable in seven patients. The survey of VZV-related symptoms revealed five additional possible VZV-cases. In cases where suitable plasma samples were available (10 out of 12), VZV-DNA was present almost simultaneously with the first clinical manifestations. No evidence of a preceding phase detectable by VZV-DNA only could be observed. Treatment with aciclovir was associated with a prompt reduction of VZV-DNA load. Detection of VZV-DNA in plasma in allo-SCT recipients accurately reflected the clinical presentation of VZV-infection and treatment with aciclovir. VZV-DNA detection in plasma of allo-SCT recipients appears clinically relevant as this may support early recognition and therapeutic management of VZV-infections following allo-SCT.

Detection and Toxin Typing of Clostridium perfringens in Formalin-Fixed, Paraffin-Embedded Tissue Samples by PCR

Since current microbiology methods are not suitable to detect Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples, we developed a PCR assay to detect toxin-encoding genes and the 16S rRNA gene of C. perfringens. We successfully detected and genotyped C. perfringens in tissue sections from two autopsy cases.

Role of Epstein-Barr virus DNA measurement in plasma in the clinical management of nasopharyngeal carcinoma in a low risk area

Objective: To evaluate the role of quantitative measurement of Epstein-Barr virus (EBV) DNA in the clinical management of nasopharyngeal carcinoma (NPC) in a low tumour risk area (western Europe). Methods: 22 consecutive Dutch NPC patients (11 europid) were studied. EBV DNA load in pretreatment and post-treatment plasma samples was determined. Three patients were also sampled at frequent intervals during treatment. RNA in situ hybridisation for the detection of EBV encoded RNAs (EBERs) was carried out on tumour biopsies of all cases. Results: All patients with EBER positive NPC (20/22) showed a positive EBV DNA load in plasma at the time of diagnosis (median EBV DNA level, 4.1 log10 copies/ml). Patients with EBER negative NPC had no detectable EBV DNA in plasma. After treatment, complete remission was achieved in all cases and concurrently EBV DNA in plasma became undetectable in all patients. In the three longitudinally evaluated cases, EBV DNA load gradually declined towards undetectable levels within three weeks after start of treatment. Two patients developed a distant metastasis with concomitant increases in EBV viral load. In addition, one EBER positive patient developed an EBER negative metastasis in the neck during follow up and in this case EBV DNA load remained undetectable at the time of recurrence. Conclusions: Plasma EBV DNA load measurement appears to be useful in a low tumour risk area. However, development of local recurrences may not always coincide with raised levels of EBV DNA.

Varicella zoster virus (VZV)-related progressive outer retinal necrosis (PORN) after allogeneic stem cell transplantation.

Varicella zoster virus (VZV)-related progressive outer retinal necrosis (PORN) after allogeneic stem cell transplantation

Comparable incidence and severity of cytomegalovirus infections following T cell-depleted allogeneic stem cell transplantation preceded by reduced intensity or myeloablative conditioning

Reports on infectious complications following reduced intensity conditioning (RIC) before allogeneic stem cell transplantation (allo-SCT) are equivocal. This prospective follow-up study compared the impact of cytomegalovirus (CMV) infections following RIC with fludarabine, ATG and busulphan or conventional myeloablative conditioning (MAC). Forty-eight RIC and 59 MAC patients were enrolled. The occurrence and severity of CMV infections within 100 days following allo-SCT were assessed, using plasma CMV DNA load kinetics. CMV DNAemia was observed in 21 RIC (60%) and in 19 MAC (44%) patients at risk for CMV. The mean CMV DNAemia free survival time was comparable following RIC and MAC: 70 days (95% (confidence interval) CI: 59–80 days) and 77 days (95% CI: 68–86 days), respectively (P=0.24). Parameters indicative for the level of CMV reactivation, including the area under the curve of CMV DNA load over time as well as the onset, the peak values and duration of CMV infection episodes, the numbers and duration of CMV treatment episodes and recurrent infections, were not different in both groups. During follow-up, none of the patients developed CMV disease. RIC with fludarabine, ATG and busulphan demonstrated safety comparable to conventional MAC with regard to frequency and severity of CMV infections within 100 days following T cell-depleted allo-SCT.