Indian Journal of Transplantation

: 2020  |  Volume : 14  |  Issue : 4  |  Page : 293--297

Comparison of the CMV antigenemia and CMV-DNA QPCR results in haematopoetic stem cells transplanted recipients - A retrospective observational study

Habib Ksouri, Yosra Chebbi, Anis Raddaoui, Wafa Achour 
 Laboratory, National Bone Marrow Transplant Center, Tunis, Tunisia

Correspondence Address:
Dr. Habib Ksouri
Laboratory, National Bone Marrow Transplant Center: 2, Rue Jebel Lakhdar, Tunis 1006


Objectives: Diagnosis of active cytomegalovirus (CMV) infection in hematopoietic stem cell (HSC)-transplanted patients is essential because of the serious complications it causes, especially CMV disease. Such diagnosis allows the initiation of a preemptive antiviral therapy, which avoids the development of such disease. In this retrospective study made as a part of routine work for transplanted patients, we compared two diagnosis testing for CMV-active infection such as pp65 CMV antigenemia and a quantitative real-time polymerase chain reaction (QPCR). Our purpose was to assess the contribution of these tools to the diagnosis and monitoring of active CMV infections. Materials and Methods: Five hundred and fifty-eight and 156 samples belonging to 24 HSC transplanted patients were, respectively, tested by antigenemia and QPCR. Results: For the 156 samples analyzed by both testing, a number of positive samples by a QPCR were higher than that by antigenemia (44.8% vs. 13%), with a statistically significant difference (P < 0.0001). A statistically significant correlation between viral loads of these two testing was also found (rs = 0.765). In median, QPCR becomes positive 14 days before antigenemia (P < 0.0001) and its positivity lasts for 91 days, whereas antigenemia positivity lasts only for 56 days (P < 0.001). No statistically significant difference was found between the results of these two testing in CMV disease cases. Conclusions: QPCR is a rapid, standardized assay, which permits a precocious detection of CMV DNA. Kinetic of DNA evolution is a reliable diagnostic tool and more effective than antigenemia-based assays in monitoring CMV infection.

How to cite this article:
Ksouri H, Chebbi Y, Raddaoui A, Achour W. Comparison of the CMV antigenemia and CMV-DNA QPCR results in haematopoetic stem cells transplanted recipients - A retrospective observational study.Indian J Transplant 2020;14:293-297

How to cite this URL:
Ksouri H, Chebbi Y, Raddaoui A, Achour W. Comparison of the CMV antigenemia and CMV-DNA QPCR results in haematopoetic stem cells transplanted recipients - A retrospective observational study. Indian J Transplant [serial online] 2020 [cited 2021 Mar 7 ];14:293-297
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Active cytomegalovirus (CMV) infection diagnosis in hematopoietic stem cell (HSC)-transplanted patients is essential because of the serious complications it causes. Diagnosis methods of virological markers of such infection are based mainly on the research of viral antigens on leukocytes (pp65 CMV antigenemia) and viral nucleic acids (polymerase chain reaction [PCR]).[1],[2]

During this retrospective study made as a part of laboratory routine work, we propose to evaluate the contribution of pp65 CMV antigenemia performed on circulating leukocytes and a quantitative real-time PCR (QPCR) for CMV genome detection performed on whole blood.

Samples were taken from HSC-transplanted patients for various hematological malignancies in the National Bone Marrow Transplantation Center.

 Materials and Methods


Whole blood collected from 24 patients (9 women and 15 men) hospitalized at the National Bone Marrow Transplantation Center for various hematological malignancies, with a median age of 32 (5–50 years) and all benefited from genoidentical autografts or allografts HSC.

All patients benefited from a conditioning regimen, according to their disease and had in post-transplant an immunosuppressive treatment based on cyclosporine and corticosteroids when they presented a graft-versus-host disease (GVHD)

Therapeutic strategy against cytomegalovirus

All patients were given weekly prophylactic acyclovir therapy from J5 to J21 post-transplant (intravenous acyclovir: 3 × 500 mg/m2/day for 3 weeks, followed by a maintenance phase with oral acyclovir 3 × 800 mg/m2/day, until J100 post-transplant). Patients with confirmed, active CMV infection (confirmed by CMV antigenemia and/or CMV-positive QPCR) were given a preemptive intravenous ganciclovir induction phase (2 × 5 mg/kg/day until J14 post-transplant) if neutrophil count exceeded 1 G/L. A similar oral regimen of ganciclovir therapy (10 mg/kg/day) was maintained until active CMV infection disappeared. This therapy was also maintained until J100 posttransplant if there was corticosteroid treatment for GVHD. Intravenous foscarnet (180 mg/kg/day) with the same protocol was administered when neutrophil count is <1 G/L.

Definition of active cytomegalovirus infection

Any positive pp65 antigenemia (from a single positive cell/2 × 105 cells) and/or QPCR positive were considered to be evidence of active CMV infection.

Definition of cytomegalovirus disease

CMV disease was defined as an association between compatible signs and symptoms (e.g., dyspnea, hypoxia, fever of unknown origin, malaise, or diarrhea) and detection of CMV (with at least 2000 CMV genome copies/mL) by a quantitative hybrid capture CMV assay in a tissue biopsy specimen.

Diagnosis of cytomegalovirus disease

Quantitative hybrid capture for CMV was done with a hybrid capture CMV DNA assay kit (version 2.0; Digene Corporation, Gaithersburg, MD). Specimens were processed according to the manufacturer's instructions. This assay was used to confirm the diagnosis of CMV disease.


Antigenemia tests were carried out from J21 to J100 post-transplant and extended after as long as the result remains positive. For CMV DNA QPCR, this test is performed when neutrophil count is <0.5 G/L, when there is a discrepancy between the clinical and biological picture and the result of antigenemia, and when we suspect CMV gastrointestinal disease, in which as known antigenemia is often negative.

Cytomegalovirus antigenemia: “Research on circulating leukocytes”

The search for antigenemia pp65 was performed for all patients with neutrophil count >0.5 G/L, within 2 h of sampling as recommended by the manufacturer (CINAkit, Argene Biosoft).

Quantitative real-time polymerase chain reaction: Genome research on whole blood “cytomegalovirus R-gene1 real-time polymerase chain reaction”

The blood sample is taken from a 5 ml tube containing an anticoagulant (ethylenediaminetetraacetic acid). After homogenization, a 350 μl aliquot is frozen at −80°C. DNA is extracted from this aliquot on the EZ1 automaton (Qiagen) with the kit EZ1 DNA Blood 350 μl Kit according to the manufacturer's instructions.

The 200 μl of extract obtained is frozen before carrying out the PCR. CMV QPCR was performed with CMV reagent R-gene1 (Argene Biosoft); it is carried out under a volume of 25 μl with 1 μml of cycler-ABI 70001 extract (Applied Biosystem). The results are expressed in copies per milliliter from a curve established with the included standards in the reagent.

Statistical analysis

The Chi-square test, the Yates-adjusted Chi-square test for paired series, and Spearman's correlation coefficient were, respectively, used to compare assay results' distribution according to CMV disease occurrence and to compare results and correlation between viral loads of the two assays. The student's t-test for paired series was used to compare the onset of active CMV infection with respect to the transplantation and the positivity duration of this infection reported by these two assays ([α = 5%]; “confidence interval” = 95%). SPSS software (Statistical Product and Services Solutions, version 18.0, SPSS Inc, Chicago, IL, USA) was used for statistical analyses.

Declaration of patient consent

The patient consent has been taken for participation in the study and for publication of clinical details and images. Patients understand that the names, initials would not be published, and all standard protocols will be followed to conceal their identity.

Ethics statement

Good Clinical Practice guidelines were followed. As it was a data analysis study EC approval was not deemed necessary. Study was carried out as per Declaration of Helsinki.


Frequency of active cytomegalovirus infections

All the patients benefited from 558 pp65 CMV antigenemia tests (average: 23 antigenemia/patient, extreme [5–31]). The patients also benefited from 156 CMV QPCR (average: 6 QPCR/patient, extreme [1–9]). Thirteen percent of the antigenemias were positive (median duration of positivity = 8 weeks, interval [1–14]) and 44.8% of the QPCR were positive (median duration of positivity = 13 weeks, interval [1–17]) [Table 1] and [Figure 1].{Table 1}{Figure 1}

Correlations between pp65 cytomegalovirus antigenemia and cytomegalovirus quantitative real-time polymerase chain reaction

Correlation between the qualitative results of the testing

For the 156 samples analyzed by both antigenemia and QPCR, the difference between the results of the two tests was statistically very significant (P < 0.0001) [Table 2].{Table 2}

Correlation between the quantitative results of the testing

The Spearman's correlation test found a positive relationship between the viral loads of these two testing (P < 0.0001), with a Spearman coefficient (rs = 0.765), indicating a parallel evolution of those loads, which means a correlation between the quantitative results provided by these two testing [Figure 2].{Figure 2}

The distribution of the results in three groups, respectively, according to whether the antigenemia pp65 was negative, between 1 and 9 cells positive/2 × 105 cells and >10 cells/2 × 105 cells, found a significant correlation between the results of these two testing (P < 0.05) only in the second group [Figure 3].{Figure 3}

Correlations between onset and duration of testing positivity

The median start of active CMV infection was 21-day posttransplant, extreme (7–91), which corresponds to the median values for QPCR conversion. Antigenemia becomes positive in median 14 days after QPCR, extreme (14–119) [Figure 1].

There was a statistically significant difference between the positivity start of QPCR and that of antigenemia (t = 6.31, P < 0.00001). This difference was also statistically significant between the duration of QPCR positivity and the duration of antigenemia positivity (t = 3.9, P < 0.001).

Correlations between active cytomegalovirus infection and cytomegalovirus disease

Four patients (16.6%) developed CMV disease, three had CMV gastrointestinal disease, and one had CMV interstitial pneumonia. The onset of CMV disease occurred at a median of 277-day posttransplantation (range: 110–295 days). No statistically significant difference was found between the frequencies of QPCR positivity and antigenemia positivity in the event of CMV disease [Table 3].{Table 3}


Quantification of CMV DNA by real-time PCR (QPCR) is increasingly supplanting antigenemia for the diagnosis of active CMV infection and for the evaluation of antiviral therapy after hematopoietic stem cell transplantation.[3],[4],[5],[6] This assay is more sensitive, easier and allows the detection of active CMV infection even in the case of neutropenia.[7]

In this retrospective study, we compared these two techniques to evaluate their contributions for the diagnosis and follow-up of active CMV infection.

During our work, the percentage of positive QPCRs was higher than that of the positive antigenemia (44.8% vs. 13%) [Table 1], which reflects the greater sensitivity of the QPCR in the diagnosis of an active CMV infection in HSC-transplanted recipients.[5],[7]

For the 156 samples analyzed simultaneously by these two testing, the difference between the results is statistically significant in favor of PCR (P < 0.0001) [Table 2]. As reported, the extreme sensitivity of real-time PCR means that this technique can detect the presence of small amounts of the CMV viral genome below the viral phosphoprotein detection power by the antigenemia.[4],[8]

For the comparison of QPCR viral loads with antigenemia loads, the Spearman's test is highly significant (rs = 0.765, P < 0.0001). This fact testifies the existence of a correlation between the quantifications found by these two techniques [Figure 2], as reported by some authors; indeed, among the different quantitative diagnostic techniques of active CMV infection, only QPCR seems to correlate well with antigenemia.[4],[7],[8],[9]

The distribution of patients' results in three groups, according to the quantification of antigenemia [Figure 3], found a significant correlation between the results of these two testing (P < 0.05) only for the group where antigenemia values are quite weak (1–<10 positive cells/2.105). Although the sampling is small, this fact would reflect the extreme sensitivity of the QPCR, which beyond a certain threshold of positivity, exceeds in quantitative precision of the antigenemia, which remains definitively a semi-quantitative technique.[9],[10]

QPCR becomes positive in median 14 days before antigenemia, with a median positivity of 91 days compared to 56 days for antigenemia. Correlations between onset and duration of positivity of QPCR and antigenemia show, respectively, statistically significant differences (P < 0.001 and P < 0.00001) [Figure 1]. QPCR allows the earlier detection of CMV than antigenemia because of its precision in the quantification of viral DNA, as reported.[5],[9]

QPCR testing allows better monitoring of CMV-active infection evolution and therefore a better monitoring of antiviral therapy.[3],[5]

Limitation of the study

It was a single centre study. Multicentric Studies with larger number of patients will be helpful.


QPCR is a fast, standardized assay that allows the earlier detection of CMV compared to CMV pp65 antigenemia. Its ease of implementation and its rapid result yield make it a testing of choice for the diagnosis of CMV-active infection in hematopoietic stem cell-transplanted recipients. In addition, the evolution kinetics of viral DNA is a good alternative to antigenemia for the monitoring of active CMV infection once the antiviral treatment instituted.

Financial support and sponsorship

This work was made as a part of routine work for transplanted patients.

Conflicts of interest

There are no conflicts of interest.


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