Whole blood real-time quantitative PCR for cytomegalovirus infection follow-up in transplant recipients

Garrigue, Isabelle; Boucher, Sébastien; Couzi, Lionel; Caumont, Anne; Dromer, C.; Néau-Cransac, Martine; Tabrizi, Reza; Schrive, Marie-Hélène; Fleury, Hervé; Lafon, Marie‐Edith

doi:10.1016/j.jcv.2006.01.002

Cited by 52 publications

(50 citation statements)

References 13 publications

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“…The threshold of 4 log 10 copies/ml has been proposed in the setting of SCT for initiation of pre-emptive therapy for CMV infections [Verkruyse et al, 2006], and thus corresponds to the threshold used in this study (3.6 log 10 copies/10 6 PBLs). The equivalence observed between the two ways of expressing viral load is in accordance with published studies on CMV viral loads in the setting of solid organ transplantation [Mengelle et al, 2003b;Garrigue et al, 2006]. In four leucopenic allogeneic stem cell transplanted patients, cellular normalization of CMV DNA loads permitted to initiate pre-emptive therapy earlier than if it had been expressed per ml of whole blood.…”

Section: Discussionsupporting

confidence: 84%

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre‐emptive therapy

Bressollette-Bodin

Coste‐Burel

Besse

et al. 2008

Journal of Medical Virology

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Two quantitative duplex real-time PCR assays were developed for co-amplification of human albumin and cytomegalovirus (CMV) or Epstein Barr virus (EBV) genes after automated extraction on whole blood, and compared two units for expressing viral DNA loads (copies per ml of blood or per 10 6 peripheral blood leukocytes (PBLs)) on 1,138 positive samples. Both PCRs were characterized by high sensitivity, reproducibility, and linear range. Automated extraction by a MagNA Pure LC Instrument was shown to be more efficient when peripheral blood cell count was inferior to 5 Â 10 9 PBLs/L. Albumin coamplification allows the detection of PCR inhibitors and normalization of viral load according to the number of cells calculated in the sample. The two ways of expressing viral load results were highly correlated, but quantitative differences varied in relation to variations of blood cell count. As these two viruses are highly cell associated, viral loads can be underestimated in patients with leucopenia. In the setting of pre-emptive strategies during CMV infection, the units in which results are expressed can influence clinical management, as illustrated in this article.

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Section: Discussionsupporting

confidence: 84%

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre‐emptive therapy

Bressollette-Bodin

Coste‐Burel

Besse

et al. 2008

Journal of Medical Virology

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“…As previously published, both methods are suitable for the early diagnosis of active HCMV infection 21 and exhibit a statistically significant relationship between viral load assessed by PCR and pp65 positive leucocytes. 22 Our data suggest that a delay of CMV-specific T-cell immune reconstitution in high-risk patients causes frequent CMV infection and disease and that early recovery of T-cell immunity is linked to lower rates of CMV infection and disease. Similar observations were made by Gratama et al 18 who also found a protective effect and better outcome in CMV seropositive patients with CMV seropositive donors than in those with seronegative ones.…”

Section: Discussionmentioning

confidence: 67%

Patients at high risk for CMV infection and disease show delayed CD8+ T-cell immune recovery after allogeneic stem cell transplantation

Ganepola

Gentilini

Hilbers

et al. 2007

Bone Marrow Transplant

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Human cytomegalovirus (CMV) is a major cause of death after transplantation. The frequency of pp65-specific T cells was examined in 38 HLA-A2 þ stem cell recipients during the first year after transplantation. Patients were divided into four groups based on donor/recipientand dÀ/rÀ (n ¼ 5). Peripheral blood mononuclear cells were stimulated with the CMVpp65 peptide NLVPMVATV, and the specific T-cell frequency was assessed by interferon gamma (IFN-c) ELISPOT assay. Responding T cells were characterized by flow cytometry revealing a terminal differentiated effector phenotype. Surveillance of CMV infection was carried out by realtime polymerase chain reaction (n ¼ 26) or immunofluorescence (n ¼ 12). Infection was present in 7/9 dÀ/r þ high-risk patients, and CMV disease occurred exclusively in this group with delayed or absent virus-specific T-cell recovery. In contrast, 16/24 intermediate-risk patients showed CMV-specific T cells. Our data suggest that CMV infection and disease rates are elevated in high-risk patients with delayed CMV-specific T-cell immune reconstitution and lower in those with early recovery of T-cell immunity. We recommend preferring CMV seropositive donors for CMV seropositive recipients, as this should lead to durable CMV-specific T-cell responses soon after transplantation with consecutive protection from CMV disease.

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“…13,14 Previous studies demonstrated that tests on whole blood samples are more sensitive than Figure 4 Receiver-operating characteristic (ROC) curve for qPCR as a diagnostic test for CMV reactivation in HSCT patients those on plasma or leukocyte. [15][16][17] Other authors reported that tests could be carried out on blood fractions (leukocytes or plasma) and whole blood. Nevertheless, detection of CMV DNA on whole blood was found to occur earlier and measure greater amounts of CMV DNA.…”

Section: Discussionmentioning

confidence: 99%

Human cytomegalovirus detection by real-time PCR and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries

Breda

Almeida

Carstensen

et al. 2013

Pathogens and Global Health

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Aim: Cytomegalovirus (CMV) infection is one of the most common complications in patients submitted to hematopoietic stem cell transplant (HSCT). Pre-emptive therapy has been indicated in patients with laboratory evidence of CMV replication. The aims of this study were to compare real-time PCR or pp65 antigen assay methodologies to detect CMV replication in HSCT patients, define a viral load threshold for initiation of pre-emptive therapy, and assess the feasibility of its implementation in hospitals of countries with low and middle income. Material and methods: Human CMV detection by real-time PCR and pp65 antigen assay was carried out in blood and plasma samples of HSCT patients collected weekly during 3 months. Pre-emptive therapy was based on CMV antigenemia results. Results: Twenty-one patients were monitored with a total of 227 samples collected; 13 (62%) patients were children. A poor correlation was observed between qualitative results, though quantitative results showed statistically significant difference, with higher viral loads detected in patients with positive antigenemia. Compared to a positive antigenemia, a cutoff value of 1067.5 copies/ml, 3.03 log 10 /ml, for viral load was obtained with 100% sensitivity and 71% specificity. Conclusion: CMV real-time PCR in whole blood was suitable for monitoring HSCT patients. However, its high cost is a limiting factor, and it could be used to monitor selected patients, those with prolonged leukopenia and underweight children, and subsequently switched to pp65 antigen test. Further studies involving larger numbers of patients should be performed to confirm this statement.

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Whole blood real-time quantitative PCR for cytomegalovirus infection follow-up in transplant recipients

Cited by 52 publications

References 13 publications

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre‐emptive therapy

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre‐emptive therapy

Patients at high risk for CMV infection and disease show delayed CD8+ T-cell immune recovery after allogeneic stem cell transplantation

Human cytomegalovirus detection by real-time PCR and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries

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