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  • These assays determine IFN production

    2020-07-31

    These assays determine IFN-γ production after stimulation of whole blood or peripheral blood mononuclear FTI 277 HCl (PBMCs) with CMV-specific antigens, viral peptides or whole virus lysates [4]. The QuantiFERON® assay (Qiagen, Hilden, Germany) and the T-Track®-CMV ELISpot (Lophius, Regensburg, Germany) were standardized and approved for commercial use in clinical settings in Europe. Various reports have compared the performance of these assays in SOT recipients. While previous studies have shown that a higher CMV-specific CMI pre-Tx/end of prophylaxis is associated with a lower incidence of CMV reactivation/infection, they have not provided a cut-off of the CMV-specific CMI, which correlates with protection from CMV reactivation/infection [8,12,13].
    Objectives
    Study design Thirty KTx recipients were enrolled in this pilot study from December 2015 to May 2016. Patient demographics are presented in Table 1. The inclusion criteria were: age ≥ 18 years and CMV-IgG serostatus (D+/R−, D+/R+, D−/R+) pre-Tx. All patients were enrolled during the first 0–3 months post-Tx. For each patient a minimum follow-up of 3 months was considered. Based on their CMV-IgG status pre-Tx, patients were divided into two groups: pre-emptive (D−/R+, D+/R+) and prophylaxis (D+/R−). T-Track-CMV was performed at month 1 post-Tx (pre-emptive group) or end of prophylaxis and one month thereafter (prophylaxis group), concurrently with the QuantiFERON-CMV. QuantiFERON-CMV was performed every 2–4 weeks (pre-emptive) or monthly (prophylaxis), parallel to CMV-DNA quantification by PCR. CMV reactivation or primary infection was defined as CMV-DNA >780 IU/ml (pre-emptive) or >40 copies/ml equals 62.4 IU/ml (prophylaxis), regardless of symptoms. Tissue-invasive disease was defined as symptomatic CMV organ manifestation (colitis, retinitis, carditis, etc.). All patients with CMV reactivation/infection received antiviral therapy. Oral valganciclovir (adapted to the kidney function) was preferred. Intravenous ganciclovir was administered in one case of tissue-invasive disease and two cases of primary CMV infection. The consensus for starting antiviral therapy in Europe is 1000 IU/ml [14]; which is equivalent to 641 copies/ml.
    QuantiFERON and ELISpot assays Concurrently, peripheral blood was collected in 3 (each 1 ml) QuantiFERON tubes (negative control, CMV-antigen and positive control) and 9 ml blood were collected in heparin tubes for the T-Track-CMV. The QuantiFERON tubes were incubated for 19 h at 37 °C and processed according to the manufacturer’s instructions. The viral peptides in the CMV-antigen tube are mapped within pp65, IE-1, pp50, IE-2, gB and pp28 and their presentation is restricted to prevailing HLA (human leukocyte antigens) class I molecules (present in 98% of the Caucasian population) [15]. For the T-Track-CMV, PBMCs were isolated, counted and adjusted to 2 million lymphocytes per ml. 200,000 lymphocytes/well were added to a 96-well ELISpot plate and stimulated with two T-activated® CMV-proteins, IE-1 and pp65, according to the manufacturer’s instructions. The median of quadruplicate stimulations was considered for further analysis and values of negative controls were subtracted from CMV-specific values, resulting in spot forming units (SFU). The QuantiFERON-CMV results were considered positive at ≥0.2 IU/ml IFN-γ, while the T-Track-CMV was defined as positive at ≥10 SFU.