CE-IVD validation of CMV ELITe MGB assay in combination with ELITe InGenius,

an innovative sample-to-result solution for quantitative transplant pathogen monitoring

C. Bittoto, S. Costa, M. Enrietto, S. Patan, F. Gorreta, A. Estampes, C. Olivo, G. Stefanuto & W. Mahoney
ELITechGroup Molecular Diagnostics
26th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), April 9-12th Amsterdam, the Netherlands

OBJECTIVES

Clinical Performance Testing

The CMV ELITe MGB assay is a quantitative nucleic acids amplification assay for the detection and
quantification of Human Cytomegalovirus (CMV) based on MGB technology. The validation study was
performed with whole blood and plasma samples in combination with ELITe InGenius the first fully automated
sample-to-result solution introduced with a comprehensive quantitative transplant pathogen monitoring menu.

ELITe InGenius (ELITechGroup Molecular Diagnostics) automatically performs nucleic acid extraction, PCR
set-up, amplification and results analysis integrating in a single platform 12 extraction modules and 12
independently controlled unitary thermal cyclers equipped with 6 optical channels.
Nucleic acid extraction was performed in prefilled unitary cartridge, ELITe InGenius SP 200 (EMD), using a
magnetic bead technology. 200 L of samples (whole blood or plasma) were automatically dispensed in the
lysis buffer of the extraction cartridge with an internal control to check the process integrity. Nucleic acids
were eluted in 100 L in a dedicated storage tube. 20 L of extracted nucleic acid and 20 L of CMV 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.
CMV ELITe MGB kit consists of a quantitative monoreagent, based on MGB technology Real-Time PCR. The
verification and validation of CMV ELITe MGB Kit in combination with ELITe InGenius was based on:
(1) analytical studies to verify the PCR performances: efficiency, linearity, accuracy, repeatability, reproducibility;
and sensitivity; (2) analytical studies to verify the whole process (extraction and PCR) performance: linearity,
LoD, LoQ, conversion factor to International Unit with blood and plasma and reproducibility with certified
reference materials (Qnostics Ltd. and Acrometrix); (3) clinical study to evaluate the diagnostic sensitivity and
specificity assessed by testing clinical samples and negative donor samples with each matrix; (4) robustness
study: calibration stability, control chart consistency, cross contamination, whole system failure.
Sample characteristics and performance acceptance criteria are summarized in Table 1.
Performance Testing

Samples

Performance Criteria

Efficiency

3 x 100,000 gEq/reaction

Ct FAM <27 for all replicates

Linearity

CMV plasmid DNA dilution panel, consisting of 6

members from 10 to 106 gEq /reaction tested in
5 replicates

R2>0.99

Accuracy

CMV plasmid DNA at 50,000, 5,000 and 500

gEq/reaction in presence of IC tested in triplicate
with two instruments

6/6 replicates falling within

range nominal titer 0.35 Log

Intra-run repeatability

Standards tested in triplicate on the same run

with three instruments

Ct CV<2%

Inter-run repeatability

Standards tested in triplicate on three runs on

three different days with two instruments

Ct CV<3%

Reproducibility

Standard performed on six runs on two

different laboratories

Ct CV<3%

Sensitivity for target

CMV plasmid DNA at 10 copies/reaction in

presence of IC tested in nine replicates with two
instruments.

18/18 replicates
were detected positive

IC plasmid at 6,000 copies/reaction tested in

nine replicates with two instruments

18/18 replicates were detected, Ct<32

Samples

Performance Criteria

Linearity

CMV dilution panel* from 106 IU/mL to 102 IU/mL

tested in triplicates carrying out the whole analysis procedure

R2 = 0.99, CV% of CMV Ct < 3%,

log < 0.5 for each level for both matrices

LoD

CMV dilution panel* from 316 to 10 IU/mL tested

in 20 replicates carrying out the whole analysis
procedure

Probit regression analysis

CMV dilution panel* from 106 IU/mL to 10 IU/mL

tested carrying out the whole analysis procedure

Lowest or Highest concentration that gives 100%

of positiveness and 95% probability that the
quantitative results were accurate

Sensitivity for IC
Whole Process Analytical
Performance Testing

Lower and Upper LoQ

Conversion factor
calculation
Reproducibility with certified
reference materials

Dilutions of the 1st WHO IS for Human CMV for NAT in whole blood and plasma tested in 10
replicates carrying out the whole analysis procedure
QCMD 2014 panel (CMVDNA14) and CMV
Molecular Q panel (Qnostics), AcroMetrix
CMVtc panel (ThermoFisher)

Correct identification, Quantification within 0.5

Log for samples within LOQ

Performance Criteria

60 positive whole blood

and 54 positive plasma samples
58 negative whole blood
and 54 plasma samples

Clinical sensitivity
Clinical specificity

>90%

The LoD and the LoQs obtained with Whole Blood and Plasma matrix and determined in IU/ml and qEq/mL
is reported in the Table 5.

>90%

Robustness testing

Samples

Performance Criteria

Calibration stability

Standards tested in triplicate at t0, t7, t15, t30

Ct CV% <5

Control stability

Positive Control tested in 20 replicates

5 series of 6 high positive samples (at 4 Log IU/
mL) alternating with 6 negative samples
Positive sample at 750 IU/mL tested in 60 replicates

100% quantified within 2SD

Cross contamination

MATERIALS AND METHODS

Samples

Whole system failure

100% of correct identification

Limit of Detection

Whole Blood

Plasma

109 IU/mL => 151 gEq/mL

88 IU/mL => 293 gEq/mL

Lower Limit of Quantification

178 IU/mL => 247 gEq/mL

178 IU/mL => 593 gEq/mL

Upper Limit of Quantification

1,000,000 IU/mL=>1,400,000 gEq/mL

1,000,000 IU/mL => 3,500,000 gEq/mL

Table 5: LoD and LoQ results in IU/ml and qEq/mL

<2% of incorrect result

Table 1: Sample characteristics and performance acceptance criteria

*prepared by diluting the 1st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques in
CMV DNA - negative EDTA WB and Plasma

Clinical Studies
The clinical sensitivity and the clinical specificity obtained after discrepant sample analysis are presented in
the Table 6.
Whole Blood

RESULTS
CMV ELITe MGB Kit tested in combination with ELITe InGenius system passed all the performance acceptance
criteria established for the analytical and clinical studies regardless the sample matrix tested.

Analytical Studies

Plasma

Diagnostic Sensitivity

100% (60/60)

100% (54/54)

Diagnostic Specificity

93.2% (55/59)

98.3% (57/58)

Table 6: Diagnostic sensitivity and specificity results

The quantification reproducibility with CMV positive plasma samples versus the CE-IVD reference method
was assessed by regression analysis (Graph 1: Fitted Line Plot).

The results obtained with certified reference material to assess the integrated system performance are
described in Table 2, 3 and 4.

Panel
Member

Consensus
Value
Log IU/mL

Standard
Deviation

Positive /
Replicates

Mean Value
Result
Obtained
Log IU/ml

LOG
obtainedexpected
Log IU/mL

Acceptance
Criteria

CMVDNA14-01

2.468

0.343

2/2

2.256

-0.212

Passed

CMVDNA14-02

3.034

0.281

2/2

2.915

-0.119

Passed

CMVDNA14-03

3.383

0.368

2/2

3.185

-0.198

Passed

CMVDNA14-04

3.014

0.251

2/2

2.976

-0.038

Passed

CMVDNA14-05

1.859

0.462

2/2

1.706

-0.153

Passed

CMVDNA14-06

2.767

0.325

2/2

2.526

-0.241

Passed

CMVDNA14-07

4.030

0.280

2/2

3.924

-0.106

Passed

CMVDNA14-08

Negative

N.A.

0/2

Passed

CMVDNA14-09

2.065

0.512

2/2

1.273

< LoQ

Passed

CMVDNA14-10

3.947

0.278

2/2

3.946

-0.001

Passed

Graph 1: Regression analysis of CMV plasma positive samples

Graph 2: Scatter Plot of CMV plasma positive samples

A R-Sq equal to 80.6% demonstrates a good correlation between the quantification of the two methods.
The Log difference (IU/ml) between both methods is shown on the Graph 2 (Scatter plot).
All the samples, except 3, had a Log difference (IU/ml) within 0.5 Log confirming the good reproducibility
between the two methods.

Table 2: CMV DNA 2014 QCMD panel results (IU/ml)

All samples (10) were correctly detected. Eight (8) out of nine positive samples were quantified within the
range defined by the EQA Consensus 1 Standard Deviation (SD) and one positive sample (CMVDNA14-09)
had a titer below the lower limit of quantification (LLoQ).
Sample

Nominal titre Log10 Positive / Replicates

Mean results Log10

IU / mL

Acceptance Criteria

CMVMQP01-High

5.000

2/2

5.024

Passed

CMVMQP01-Medium

4.000

2/2

3.996

Passed

CMVMQP01-Low

3.000

2/2

3.060

Passed

CMVMQP01-Negative

0/2

Passed

Robustness Studies
Robustness studies had confirmed the total absence of cross-over and cross-contamination during repeated
runs and the whole system failure was assessed at 1.67%.
The stability of the standard curve has been established at 30 days since the quantification results obtained
at 30 days were comparable to the results obtained at time 0.
The Graph 3 shows the control chart verification obtained with 20 replicates of CMV ELITe Positive Control.

The 80% of the samples were quantitated

within the mean 1SD which was equal
to 0.114 Log and 100% were quantitated
within 2SD.

Table 3: CMV Molecular Q panel results

All positive samples were detected as positive with a titer within the expected value 0.5 Log.
Sample

Nominal titre Log10 Positive / Replicates

Mean results Log10

IU / mL

Acceptance Criteria

CMV DNA 3E6

6.477

2/2

6.386

Passed

CMV DNA 3E5

5.477

2/2

5.444

Passed

CMV DNA 3E4

4.477

2/2

4.473

Passed

CMV DNA 3E3

3.477

2/2

3.441

Passed

CMV DNA 3E2

2.477

2/2

2.575

Passed

Table 4: AcroMetrix CMV Panel results

All positive samples were detected as positive with a titer within the expected value 0.5 Log.

Graph 3: Control Chart of CMV Positive Control

CONCLUSION
The excellent results obtained during the verification and validation study support the CE-IVD marking of
CMV ELITe MGB assay in combination with ELITe InGenius system for the detection and the quantification
of DNA extracted and amplified from whole blood and plasma.
EMD-CMV- ECCMID2016-550-2016/00EN