Quantitative analysis of cytomegalovirus load using a real-time PCR assay

Tanaka, Naoko; Kimura, Hiroshi; Iida, Keiji; Saito, Yumiko; Tsuge, Ikuya; Yoshimi, Ayami; Matsuyama, Takaharu; Morishima, Tsuneo

doi:10.1002/(sici)1096-9071(200004)60:4<455::aid-jmv14>3.0.co;2-q

Cited by 163 publications

(146 citation statements)

References 31 publications

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“…13 Real-time PCR was performed using a TaqMan PCR Kit and Model 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA), as described previously. 9,14,15 The detection limit of this assay was 100 copies ml -1 .…”

Section: Serologymentioning

confidence: 91%

Transmission of cytomegalovirus via breast milk in extremely premature infants

et al. 2010

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Objective: We prospectively evaluated the rate of postnatal cytomegalovirus (CMV) transmission through breast milk in extremely premature infants to address the impact of CMV infection on preterm infants during lactation.Study Design: A total of 25 mothers and 27 infants (two sets of twins) with birth weights <1000 g and/or gestational ages <28 weeks were enrolled in the study. They were mostly fed frozen-thawed breast milk. Breast milk, serum and urine samples were collected every 2 weeks and screened for CMV infection using the real-time polymerase chain reaction.Result: All of the 21 CMV-seropositive mothers had detectable CMV DNA in their breast milk, with a peak at 4 to 6 weeks postpartum. CMV infection was confirmed in only one infant (4.3%) who displayed almost no clinical symptoms. Conclusion:At our institutes, we mainly use frozen-thawed breast milk. We found low CMV transmission rates even in extremely premature infants, and the CMV-positive infant did not develop serious symptoms.

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Section: Serologymentioning

confidence: 91%

Transmission of cytomegalovirus via breast milk in extremely premature infants

et al. 2010

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“…The viral loads were normalized to 10 000 human genomic equivalents obtained by quantification of the albumin gene. 25 Normalized viral load above 100 was considered clinically significant and preemptive therapy with ganciclovir was initiated. Patients were treated with i.v.…”

Section: Patients and Healthy Donorsmentioning

confidence: 99%

Signature profiles of CMV-specific T-cells in patients with CMV reactivation after hematopoietic SCT

Krol

Stuchlý

Hubáček

et al. 2010

Bone Marrow Transplant

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Depletion of cellular immunity as a consequence of conditioning before allogeneic hematopoietic SCT (HSCT) frequently results in CMV reactivation, which may in turn lead to life-threatening infections and require timely antiviral treatment. We have investigated the functional signatures of CMV-specific CD4 þ and CD8 þ T-cells in 191 samples from 118 individuals. We compared healthy donors with both patients with high and undetectable viral loads, and those who controlled and did not control their CMV reactivations. Polychromatic flowcytometric measurements of CD154 (CD40L), intracellular cytokines (IFNc, IL2) and a degranulation marker (CD107a) allowed us to assess the functional status of various T-cells simultaneously. We found that dual IFNc/ IL2-producing CD8 þ T-cells were present in patients controlling their CMV reactivations but absent from noncontrollers. CD8 þ T-cells that produce only IFNc were the most abundant subtype, but they most likely represent non-protective memory cells. Distinct functional signatures were examined by hierarchical clustering, and this revealed that, unlike polyfunctional CD8 þ T-cells, CD8 þ T-cells that produce IFNc alone were not functioning in concert with other subsets. In conclusion, our study revealed functional signatures that may be useful for immune monitoring, and it could change the interpretation of previous studies that assessed only IFNc.

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“…12,13 US17-PCR was performed in Otsuka Assay Laboratories (Otsuka Pharmaceutical, Tokyo, Japan) and IE-PCR was performed in SRL (Tokyo, Japan). Briefly, DNA extracted from 100 l of plasma was subjected to PCR using TaqMan Universal PCR Master Mix (PE Biosystems, Foster City, CA, USA) and the PCR product was detected as an increase in the fluorescent intensity using ABI Prism 7700 (PE Biosystems).…”

Section: Real-time Pcr Assaymentioning

confidence: 99%

“…10 Although it might be too sensitive for discrimination of high-risk patients for CMV disease, 11 the development of real-time PCR has made it possible to quantitatively evaluate the virus load. [12][13][14] However, the threshold of the virus load to discriminate high-risk patients has not been determined, although the threshold of the antigenemia value has been reported. 15,16 Serially monitoring the virus load without administering ganciclovir is the only method to address this issue, but it is unethical.…”

mentioning

confidence: 99%

Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after hematopoietic stem cell transplantation

Tanaka

Kanda

Kami³

et al. 2002

Bone Marrow Transplant

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Summary:We compared a CMV virus load determined by realtime PCR with an antigenemia value to analyze the correlation between these two methods. We also compared the values for virus load determined by the two distinct real-time PCR methods, which amplify the US17 region and immediate-early (IE) gene of CMV, respectively, to evaluate the reliability of these methods. Two hundred and sixty-five samples were obtained weekly from 29 patients, who had engraftment after unrelated bone marrow transplantation or HLA-mismatched related blood stem cell transplantation. CMV infection was detected in 115 samples from 22 patients by US17-PCR and 69 samples from 20 patients by the antigenemia assay. Fifty-eight samples were positive for both assays, but 57 and 11 samples were positive only for US17-PCR and antigenemia, respectively. A good correlation of the results of US17-PCR and antigenemia was demonstrated (r = 0.61). All antigenemia-positive samples and randomly selected antigenemia-negative samples were subjected to IE-PCR. The results of IE-PCR showed a good correlation with those of antigenemia (r = 0.64). Furthermore, the best correlation was observed between US17-PCR and IE-PCR (r = 0.83). In conclusion, both real-time PCR methods showed a good correlation with the antigenemia assay, and could be used to monitor CMV infection after hematopoietic stem cell transplantation.

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Quantitative analysis of cytomegalovirus load using a real-time PCR assay

Cited by 163 publications

References 31 publications

Transmission of cytomegalovirus via breast milk in extremely premature infants

Transmission of cytomegalovirus via breast milk in extremely premature infants

Signature profiles of CMV-specific T-cells in patients with CMV reactivation after hematopoietic SCT

Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after hematopoietic stem cell transplantation

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