A Real-Time Quantitative PCR Targeting the Viral Vector for the Monitoring of Patients Treated with Axicabtagene Ciloleucel.

Axicabtagene ciloleucel or axi-cel [CD19 chimeric antigen receptor (CAR) T cell] has been recently approved for refractory/relapsed diffuse large B-cell lymphoma and primary mediastinal B-cell lymphoma. Proliferation of CAR T cells after infusion and their persistence have been reported as important factors. Laboratory tools are needed for the monitoring of patients. We developed a vector-based, simple, and accurate real-time quantitative PCR (qPCR) to measure axi-cel vector copy number in peripheral blood samples. Primers and probe targeting the 5'LTR region of the gammaretroviral vector (mouse stem cell virus) were designed for amplification. To generate standard curves, mouse stem cell virus plasmid was subcultured and quantified using droplet digital PCR. The method was applied to quantify vector copy number in blood samples from patients treated with axi-cel. The limit of quantification of the qPCR assay was established at 2.2 copies/µL in DNA eluate. The qPCR method was well correlated with flow cytometry findings; however, the assay appeared to be more sensitive than flow cytometry. The kinetics observed in blood samples from treated patients were in agreement with previously reported findings. In conclusion, we developed a sensitive and accurate qPCR assay for the quantification of transgenic CAR T cells, which can be a useful additional tool for the monitoring of patients treated with axi-cel.

Donor-to-recipient transmission and reactivation in a kidney transplant recipient of an inherited chromosomally integrated HHV-6A: Evidence and outcomes.

Human herpesvirus (HHV)-6A can be inherited and chromosomally integrated (iciHHV-6A), and donor-to-recipient transmission has been reported in solid organ transplant. However, when HHV-6A reactivation happens after transplant, the source of HHV-6A is often not evident and its pathogenicity remains unclear. Here, we present an exhaustive case of donor-to-recipient transmission and reactivation of iciHHV-6A through kidney transplant. The absence of HHV-6A genome from the nails of the recipient excluded a recipient-related iciHHV-6A. Viral loads > 7 log10 copies/106 cells in donor blood samples and similarities of U38, U39, U69, and U100 viral genes between donor, recipient, and previously published iciHHV-6A strains are proof of donor-related transmission. Detection of noncoding HHV-6 snc-RNA14 using fluorescence in situ hybridization analysis and immunofluorescence staining of HHV-6A gp82/gp105 late proteins on kidney biopsies showed evidence of reactivation in the transplanted kidney. Because HHV-6A reactivation can be life threatening in immunocompromised patients, we provide several tools to help during the complete screening and diagnosis.

Enteroviruses in blood of patients with type 1 diabetes detected by integrated cell culture and reverse transcription quantitative real-time PCR.

AIMS: Enteroviruses (EV) have been associated with type 1 diabetes (T1D), but EV RNA detection has been reported in only a small proportion of T1D patients. We studied whether integrated cell culture and reverse transcription real-time PCR could improve EV detection in blood samples from patients with T1D. METHODS: Blood was collected from 13 patients with T1D. The presence of EV RNA in blood was investigated by using real-time RT-PCR. In addition, plasma and white blood cells (WBC) were inoculated to BGM and Vero cell line cultures. Culture supernatants and cells collected on day 7 and day 14 were tested for EV RNA by real-time RT-PCR. Enterovirus identification was performed through sequencing of the VP4/VP2 region. RESULTS: Enterovirus RNA was detected in blood by using real-time RT-PCR in only one out of 13 patients. The detection of EV RNA in cultures inoculated with clinical samples (plasma and/or WBC) gave positive results in five other patients. The viral loads were low, ranging from 45 to 4420 copies/ng of total RNA. One isolate was successfully identified as coxsackievirus B1. CONCLUSIONS: Integrated cell culture and reverse transcription real-time PCR can improve the detection rate of EV in blood samples of patients with T1D and can be useful to investigate further the relationship between EV and the disease.

Lopinavir/ritonavir resistance in patients infected with HIV-1: two divergent resistance pathways?

The HIV-1 protease L76V mutation has been described recently as conferring high-level resistance to lopinavir/ritonavir (LPV/r). The aim was to identify the factors and particularly protease mutations associated with the presence of L76V in treatment-experienced patients infected with HIV-1 who have failed virologically an LPV/r-based antiretroviral therapy regimen. This is a retrospective exploratory study. Patients were eligible if they were in care at the Northern France AIDS reference center between 2000 and 2009, failed virologically an LPV/r-based regimen, and infected with HIV-1 strains carrying LPV/r-resistant mutations (genotype resistance test after failure). Multivariate logistic regressions were used to compare LPV/r-resistant patients infected with virus harboring the L76V mutation or not (L76V positive/L76V negative). Twelve patients with virus L76V positive were identified and compared to 24 patients with virus L76V negative selected at random. Demographic and clinical data were not different significantly between the two groups. In univariate analyses, of the mutations found in ≥10% of patients, L89M and Q58E were more prevalent in viruses L76V positive than L76V negative (L89M, 42% vs. 0%, P = 0.0007; Q58E, 50% vs. 25%, P = 0.1). In contrast, I54V, G73S and L90M were less prevalent in viruses L76V positive than L76V negative (I54V, 42% vs. 83%, P = 0.01; G73S, 0% vs. 33%, P = 0.02; L90M, 25% vs. 83%, P = 0.0006). L90M, I54V and Q58E were associated with L76V in a multivariate analysis (P < 0.0001, P = 0.002, and P = 0.008, respectively). These results suggest two divergent pathways leading to LPV/r resistance. One contains the L76V and Q58E mutations and the other contains the L90M and I54V mutations.