Cytomegalovirus DNAemia and risk of mortality in allogeneic hematopoietic stem cell transplantation: Analysis from the Spanish Hematopoietic Transplantation and Cell Therapy Group

The net impact of cytomegalovirus (CMV) DNAemia on overall mortality (OM) and nonrelapse mortality (NRM) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a matter of debate. This was a retrospective, multicenter, noninterventional study finally including 749 patients. CMV DNA monitoring was conducted by real-time polymerase chain reaction (PCR) assays. Clinical outcomes of interest were OM and NRM through day 365 after allo-HSCT. The cumulative incidence of CMV DNAemia in this cohort was 52.6%. A total of 306 out of 382 patients with CMV DNAemia received preemptive antiviral therapy (PET). PET use for CMV DNAemia, but not the occurrence of CMV DNAemia, taken as a qualitative variable, was associated with increased OM and NRM in univariate but not in adjusted models. A subcohort analysis including patients monitored by the COBAS Ampliprep/COBAS Taqman CMV Test showed that OM and NRM were comparable in patients in whom either low or high plasma CMV DNA threshold (<500 vs ≥500 IU/mL) was used for PET initiation. In conclusion, CMV DNAemia was not associated with increased OM and NRM in allo-HSCT recipients. The potential impact of PET use on mortality was not proven but merits further research.     

Increased incidence and unusual presentations of CMV disease in kidney transplant recipients after conversion to belatacept

Belatacept may increase cytomegalovirus (CMV) disease risk after conversion from CNI-based therapy. We analyzed CMV disease characteristics after belatacept conversion. Propensity score matching was used to compare CMV disease incidence in belatacept- and CNI-treated kidney transplant recipients (KTRs). CMV disease characteristics and risk factors under belatacept were analyzed. In total, 223 KTRs (median age [IQR] 59.2 years [45.4–68.5]) were converted to belatacept (median of 11.5 months [2.5–37.0] post-transplantation); 40/223 (17.9%) developed CMV disease. Independent risk factors included increased age (p = .0164), D+/R− CMV serostatus (p = .0220), and low eGFR at conversion (p = .0355). Among 181 belatacept-treated patients matched to 181 controls, 32/181 (17.7%) experienced CMV disease (vs. 5/181 controls [2.8%]). CMV disease cumulative incidences were 6.33 and 0.91/100 person-years (p-y) in belatacept and control groups, respectively. CMV disease risk was particularly high in elderly patients (converted >70 years) and those with eGFR <30 ml/min; cumulative incidences were 18.4 and 5.2/100 p-y, respectively. CMV diseases under belatacept were atypical, with late-onset disease (24/40 patients [60%]), high CMV seropositivity (27/40, 67%), increased severe and tissue-invasive disease rates (gastrointestinal involvement in 32/40 [80%]) and life-threatening diseases (4/40 [10%]). These findings should stimulate further research to secure the use of belatacept as a valuable rescue therapy in KTRs.     

Letermovir in lung transplant recipients with cytomegalovirus infection: A retrospective observational study

Letermovir is a new antiviral drug approved for the prophylaxis of CMV infection in allogeneic stem cell transplants. The aim of the study was to assess the therapeutic efficacy of letermovir in difficult to treat CMV infections in lung transplant recipients. All lung transplant recipients between March 2018 and August 2020, who have been treated with letermovir for ganciclovir-resistant or refractory CMV infection were included in the study and analysed retrospectively. In total, 28 patients were identified. CMV disease was present in 15 patients (53.6%). In 23 patients (82.1%), rapid response was noticed, and CMV-viral load could be significantly decreased (>1 log₁₀) after a median of 17 [14–27] days and cleared subsequently in all of these patients. Five patients (17.9%) were classified as non-responder. Thereof, development of a mutation of the CMV UL56 terminase (UL-56-Gen: C325Y) conferring letermovir resistance could be observed in three patients (60%). Common side effects were mild and mostly of gastrointestinal nature. Mild adjustments of the immunosuppressive drugs were mandatory upon treatment initiation with letermovir. In addition to other interventions, letermovir was effective in difficult to treat CMV infections in lung transplant recipients. However, in patients with treatment failure mutation conferring letermovir, resistance should be taken into account.     

Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell-free cytomegalovirus

Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and "in-house" assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV-specific and MCMV-specific enzyme-labelled probes and automated chemiluminescence detection. Log-transformed HCMV-to-MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV-spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be > or = 4 log(10). A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity.     

Antigenemia,immunoblotting, and enzyme immunoassay for early diagnosis of cytomegalovirus infection in renal transplant patients

Timely and rapid diagnosis of cytomegalovirus (CMV) infection is important for the management of transplant patients. We compared three serological assays, IgM immunoblot and IgG/IgM enzyme immunoassay (EIA), as well as the detection of CMV antigens in polymorphonuclear blood leukocytes (antigenemia), for their value in the early diagnosis of CMV infection. Thirty-one patients were monitored longitudinally for 3 months after renal transplantation. Laboratory documented CMV infection occurred in 20 patients. All of these cases showed a positive IgM immunoblot result that was confirmed by at least one of the other test assays (IgG EIA 19/20, antigenemia assay 13/20, and IgM EIA 12/20). All of the ten patients whose clinical picture was compatible with symptomatic CMV disease were positive for CMV infection according to IgM immunoblot and IgG EIA, nine were positive according to the antigenemia assay, and seven were positive according to IgM EIA. With reference to the temporal pattern, the antigenemia assay indicated CMV infection significantly earlier than the serological tests (P0.05). In symptomatic patients CMV antigen-positive leukocytes were, on the average, detected on the day of onset of symptoms, whereas detection by IgM immunoblot, IgG EIA, and IgM EIA followed 8, 13, and 14 days later, respectively. These results show that: (1) the CMV antigenemia assay is very useful for the early diagnosis of symptomatic CMV infections; (2) CMV antibodies, as an indicator of CMV infection, are detectable earlier and more frequently by IgM immunoblot than by IgG/IgM EIA; (3) compared to CMV anti-genemia, the IgM immunoblot indicated CMV infection more often but significantly later; and (4) only a combination of several diagnostic methods allows optimal detection of CMV infections in renal transplant patients.     

原位ELISA法在抗巨细胞病毒药物筛选中的应用

建立一种体外筛选抗ＣＭＶ药物的ｉｓＥＬＩＳＡ法,方法 采用ＥＬＩＳＡ测定后培养细胞上的病毒抗原,并与空斑减少试验（ＰＲＡ）法进行比较。结果 ｉｓＥＬＩＳＡ法病毒接种量范围宽,重复性好,结果客观可靠,操作简便,保需５ｄ即可观察结果。结论：ＥＬＩＳＡ可代替ＰＲＡ法用于体外抗ＣＭＶ药物的筛选。     

Herpesvirus Replication Compartments: Dynamic Biomolecular Condensates?

Recent progress has provided clear evidence that many RNA-viruses form cytoplasmic biomolecular condensates mediated by liquid–liquid phase separation to facilitate their replication. In contrast, seemingly contradictory data exist for herpesviruses, which replicate their DNA genomes in nuclear membrane-less replication compartments (RCs). Here, we review the current literature and comment on nuclear condensate formation by herpesviruses, specifically with regard to RC formation. Based on data obtained with human cytomegalovirus (human herpesvirus 5), we propose that liquid and homogenous early RCs convert into more heterogeneous RCs with complex properties over the course of infection. We highlight how the advent of DNA replication leads to the maturation of these biomolecular condensates, likely by adding an additional DNA scaffold.