Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV) infections are common complications after transplantation, resulting in significant morbidity, graft loss, and occasional mortality. CMV infection is an important cause of morbidity and mortality in solid organ transplant patients, with up to 75% of patients developing or reactivating CMV infection after transplantation [1]. EBV infection is principally responsible for infectious mononucleosis, and also plays role in the etiology of various lymphomas and post-transplantation lymphoproliferative disease [2-4]. Especially in the bone marrow or solid organ transplanted, or in otherwise severely immune-suppressed patients, prolonged EBV primary infection or EBV reactivation from latency may be a serious and life-threatening complication [5,6]. BKV infection is a relatively common and early post-transplant complication after kidney transplantation [7,8]. Careful screening can prevent allograft loss and should be employed. Moreover, there is still no consensus on the ideal method for performing quantitative real-time polymerase chain reaction (PCR) in case of CMV, EBV, and BKV detection and quantification, and this increases the variability between laboratories and between studies [9]. Different detection methods are available commercially.

Therefore, accurate detection of viral DNA are of importance for the diagnosis of serious these viral disease and monitoring. ELITe InGenius (ELITech Group, Milano, Italy) is a fully automated cassette-based sample-to-result system featuring a universal extraction with multiple and independent real-time PCR enabling the user to run custom panels with mixed parameters. The validation studies in combination with CMV, EBV, and BKV are sparse. In this study, we evaluated the performance of the fully automated cassette-based real-time PCR method for CMV, EBV, and BKV in comparison with the other commercially available real-time PCR method.

The ELITe InGenius system automatically performs nucleic acid extraction, PCR set-up, PCR amplification and results analysis. In brief, nucleic acid extraction was performed in prefilled unitary cartridge, using a magnetic bead technology. The samples were automatically dispensed in the lysis buffer of the extraction cartridge with an internal control to check the process integrity. Nucleic acids were eluted. Extracted nucleic acid and ELITe MGB Kit Real-Time PCR reagent were then automatically dispensed in a unitary PCR vessel. ELITe InGenius PCR Cassette which is automatically capped by the system prior the PCR reaction. Extraction parameters, thermal profiles information and result interpretation rules are all included in a specific pre-programmed assay protocol. Precision calculations were based on the results of five technical replicate reactions performed on each of 5 consecutive days. Linearity was evaluated by using of Molecular Qnostics CMV, EBV, and BKV panel (Randox, Crumlin, UK), running each dilution in duplicates. Panels ranged in concentration from 3.457 log10 copies/mL to 5.501 log10 copies/mL, from 2.943 log10 copies/mL to 4.901 log10 copies/mL, and from 3.444 log10 copies/mL to 5.203 log10 copies/mL, for CMV, EBV, and BKV, respectively. Linear regression analysis was performed and correlation coefficient calculated. For the limit of detection (LOD) analysis (EBV and BKV), we tested ten times using patient samples having around lower limits of detection with each viral concentration. Eighteen whole blood and 2 urine samples collected from patients were processed by the CMV R-gene PCR kit (bioMérieux, Marcy l’Etoile, France) and the CMV ELITe MGB kit (ELITech Group). Thirteen whole blood and three urine samples collected from patients with four reference materials were tested by the EBV R-gene PCR kit (bioMérieux) and the EBV ELITe MGB kit (ELITech Group). Thirteen whole blood and three urine samples collected from patients with four reference materials were tested by the BK Real-time PCR kit (SML GENETREE, Seoul, Korea) and the EBV ELITe MGB kit (ELITech Group).

The assay variability of cycle threshold measurements was ≤1% for all concentrations. Total precision was 2.5%, 2.9%, and 2.8% (low control) and 0.7%, 3.1%, and 0.6% (high control) as well for CMV, EBV, and BKV assay, respectively. Analytical sensitivity of the assay was evaluated using purified control genomic DNA of known concentration, the coefficient of correlation was 0.999 (Fig. 1). LOD results were 36 copies/mL and 165 copies/mL for EBV and BKV assays, respectively. When comparing the results of 20 CMV infected samples, two samples (10%) resulted negative and 18 (90%) positive with a quantitative result for both assays and the coefficient of correlation was 0.785. When comparing the results of 16 EBV infected samples, 11 samples (68.7%) resulted negative and 5 (31.3%) positive with a quantitative result for the EBV ELITe MGB assay and only one sample (6.3%) showed positive and 15 (93.7%) negative with a quantitative result for the EBV R-gene assay. The results of four reference materials were concordant. The coefficient of correlation was 0.994. Four samples (9.6%) were positive with a quantitative result with the EBV ELITe MGB assay and negative with the EBV R-gene assay. No samples were positive with the EBV R-gene assay and negative with the EBV ELITe MGB assay. The EBV ELITe MGB assay could detect lower amount of DNA compared with the other. When comparing the results of 16 BKV infected samples, 13 samples (81.3%) resulted negative and 3 (18.7%) positive with a quantitative result for the EBV ELITe MGB assay. The results of four reference materials were concordant. There was no samples that showed positive results in the BK real-time PCR kit (SML GENETREE). The coefficient of correlation was 0.999 (Fig. 2). The BKV ELITe MGB assay showed higher sensitivity in samples containing lower amount of DNA.

Figure 1. Linearity for the ELITe MGB assay. (A) Cytomegalovirus (CMV), (B) Epstein-Barr virus (EBV), and (C) BK virus (BKV). To evaluate the linearity, each member of the Molecular Qnostics Panel was tested 2 times in the same experiment with the ELITe MGB assay (ELITech Group, Milano, Italy), and the measured values were compared to the expected ones.

Figure 2. Comparative studies of the ELITe MGB assay and the other real-time polymerase chain reaction assay. (A) Cytomegalovirus (CMV), (B) Epstein-Barr virus (EBV), and (C) BK virus (BKV). The assays used were from the following companies: ELITe MGB assay (ELITech Group, Milano, Italy) and SML (SML GENETREE, Seoul, Korea).

Over the years, quantification of viral load in blood, mostly by real-time PCR, has become a mainstay of clinical management. The reliability and accuracy of viral load determination are therefore critical for the management of transplant patients.

A study that compare the CMV ELITe MGB assay with the Abbott real-time CMV PCR Kit (Abbott Molecular Inc., Des Plaines, IL, USA) and revealed that assays showed a good correlation between CMV-DNA levels detected (r=0.856, P<0.0001). But, Abbott showed higher sensitivity in samples containing lower amount of DNA [9]. Another study compared the Cobas AmpliPrep/Cobas TaqMan (Roche Diagnostics, Branchburg, NJ, USA) platform on plasma specimens with the CMV ELITe MGB assay in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good (r=0.9575, P<0.0001) [10]. To improve standardization of diagnostic procedures for microbiological monitoring of solid organs and stem cell transplant recipients, studies for measuring variability in viral DNA quantification were needed. One multicenter study aimed to measure variability in CMV, EBV, and BKV DNA quantification, has already been performed, showed good performance of the ELITe MGB assay with a variation coefficient comprised within 5% [11].

To be effective as a diagnostic and viral load monitoring test, a NAAT assay must quantitate viral results across a wide dynamic range, be sensitive, specific, accurate and precise. In this study, the performance of the ELITe MGB assay using the ELITe InGenius system was evaluated. The turnaround time for ELITe MGB assay is only 150 minutes.

This, fully automated sample-to-result solution introduced with a comprehensive quantitative transplant pathogen monitoring menu, on various specimen types showed good variability and sensitivity. Moreover, the fully automated system, reducing demands for skilled personnel and the possibility of human error.

There are some possible limitations. The restricted spectrum of samples analyzed, which was due to the limited patient specimens obtained from a clinical molecular laboratory. Thus, more studies will be required especially EBV and BKV. Specificity is important to avoid false-positive results when diagnosing patients with viral infections. But we did not evaluate specificity and influence of interference materials.

In conclusion, the assay showed a good correlation between CMV, EBV, and BKV DNA levels detected by the routine PCR assay and the excellent validation results. Moreover, the EBV and BKV ELITe MGB assay showed higher sensitivity in samples containing lower amount of DNA. Therefore, the use of ELITe MGB assay in combination with the ELITe InGenius system allows rapid, sensitive, and reliable detection and the quantification of viral DNA, making early information-based intervention possible. But the clinical value of this assay requires further investigation.