br Objectives br Study design Thirty

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 Illumina 384 (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.
CMV status and viral loads
Statistics
Results During the first 6 months post-Tx, 12/21 (57%) pre-emptively monitored and 5/9 prophylaxis (56%) patients, developed CMV viremia (Table 1). One patient had CMV tissue-invasive disease (Fig. 1b, patient 6) and three patients developed primary infection/reactivation during antiviral prophylaxis.
Discussion Two CMV-IgG positive patients had undetectable CMI with one of the tests. Negative CMI results may be due to uncommon HLA-antigens or host inability to recognize the pp65 peptide [16]. However, in our study these two CMV R + patients had common HLA-antigens (e.g., HLA-A*24, B*18, C*07, DRB1*04, DRB1*11, DQB1*03). One of them had negative QuantiFERON-CMV values throughout the follow up without CMV reactivation (patient 5, Fig. 1). It could therefore be considered as false negative. The other patient (patient 17, Fig. 1) had negative T-Track-CMV values but positive QuantiFERON-CMV values and developed CMV reactivation. Thus, in this patient positivity to the QuantiFERON-CMV did not indicate protection from reactivation. The negative T-Track-CMV correctly identified the patient as susceptible to CMV reactivation. In a cohort of haemodialysis patients, the agreement between Quantiferon-CMV and CMV-IgG was lower than in our study, the agreement between T-Track-CMV and serology comparable to our results. The discrepancy could be caused by a lower number of patients in our study or the use of different serological assays [17].