Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures

Hamprecht, Klaus; Mikeler, Elfriede; Jahn, Gerhard

doi:10.1016/s0166-0934(97)00148-1

Cited by 26 publications

(18 citation statements)

References 25 publications

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“…DNA was extracted from saliva and DBS samples from the newborn preterm infants; these samples and breast milk were processed as described previously, and DNA was extracted by the method of phenolchloroform [16,17].…”

Section: Dna Extractionmentioning

confidence: 99%

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Detection and gB genotyping of CMV in Mexican preterm infants in the context of maternal seropositivity

Arellano‐Galindo

Villanueva-García

Cruz-Ramirez

et al. 2014

J Infect Dev Ctries

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Introduction: Congenital (CI) and perinatal cytomegalovirus (CMV) infections (PI) can be linked to maternal CMV seropositivity, with fatal consequences in preterm newborns. GB genotyping has been used to analyze genotypic similarity in mothers and infants. The frequency of CMV infection in the context of maternal seropositivity and the viral gB genotypes as well as the genotypic similarity in mothers and preterm infants were investigated. Methodology: Saliva samples and dry blood spots (DBS) were taken weekly from preterm newborns from birth until the first month of life, and breast milk samples were taken from their mothers weekly during the first month of lactation. CMV IgG seroprevalence of the mothers and CI or PI in the infants were established. The gB status and genotypic similarities were established retrospectively in DBS and in the breast milk samples. Results: In total, 387 neonates and 375 mothers were enrolled. The maternal CMV-positive IgG serology was 97.3% (365/375). Neonatal CMV was found in 5.1% (20/387) of newborns, and one infant presented with CMV-compatible symptoms. CI was 2.5% and PI in the first month after birth was 11.8%. GB2 was the most prevalent genotype and was also the genotype preferentially transmitted to newborns by mothers with mixed infections. Conclusions: CMV PI and CI in preterm infants from highly seropositive mothers was high, but the rate of symptomatic infection was low. The prevalent genotype was gB2, and this genotype was preferentially transmitted to newborns by mothers with mixed infections.

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Section: Dna Extractionmentioning

confidence: 99%

“…To corroborate the DNAemia in the neonatal DBS samples and DNAlactia in the maternal samples before genotyping, the DNA from each sample was analyzed with nested PCR using primers designed to amplify the fourth exon of the CMV ie1 gene, as described previously [17].…”

Section: Pcr Assaymentioning

confidence: 99%

Detection and gB genotyping of CMV in Mexican preterm infants in the context of maternal seropositivity

Arellano‐Galindo

Villanueva-García

Cruz-Ramirez

et al. 2014

J Infect Dev Ctries

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“…8 Semi-quantitative limiting dilution nested PCR from native plasma was performed whenever HCMV-plasma-DNAemia was detected. 9 Antiviral therapy with GCV was initiated (initiation 5 mg/kg/ twice a day, maintenance 5 mg/kg/day), when two consecutive PCRs from leukocytes were positive. GCV-resistant infection was alternatively treated with PFA (3 ϫ 40/3 ϫ 50 mg/kg/day) or CDV (5 mg/kg weekly).…”

Section: Hcmv-monitoring Antiviral Therapy and Resistance Screeningmentioning

confidence: 99%

Rapid development of ganciclovir-resistant cytomegalovirus infection in children after allogeneic stem cell transplantation in the early phase of immune cell recovery

Eckle

Lang

Prix

et al. 2002

Bone Marrow Transplant

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Summary:As recently reported, children having T cell-depleted peripheral blood stem cell transplantation (PBSCT) might be at increased risk for the development of drug resistance. To investigate if delayed immune recovery was a potential risk factor, the recovery of the CD3 + , CD4 + , CD8 + and CD19 + cells was related retrospectively to genotypic detected resistance development in three pediatric patients with ganciclovir ( Keywords: HCMV; UL97; UL54; PBSCT; immune recovery; CD34 + blood stem cells Studies in AIDS patients made clear that ganciclovir (GCV) resistance develops in a slow but progressive manner. GCV-resistant human cytomegalovirus (HCMV) seems to infect immunocompromised patients who have either profound immunosuppression with very high viral loads or who have undergone long periods of therapy. 1 Until recently, there have been only scattered reports of GCV resistance in transplant recipients. As reported, in a study of 240 allograft recipients, subsets at special risk were HCMV-seronegative patients who received an organ from an HCMV-seropositive donor and those patients who required long periods of intense immunosuppression. On average, the patients in this report were treated with GCV for a mean of 190 days, which suggested that the duration of GCV exposure is also an important factor in the development of resistance. 2 In the BMT setting, there is even less knowledge about GCV resistance. After BMT in adults there exists to date only one report about a patient with phenotypic, as well as genotypic GCV resistance, 3 whereas a recent study using RFLP analysis limited to the detection of some frequent occurring UL97 mutations (codons: 460, 520, 594, 595) did not reveal any evidence for the emergence of GCV resistance in 10 out of 34 adults treated after PBSCT. 4 However, according to our experience the rapid emergence of GCV resistance in codons 591/603 should not be ignored. 5,6 Initially, Wolf et al 7 reported on the early emergence of GCV resistance in children with primary combined immunodeficiency (CID) after T cell-depleted BMT in four out of six cases. Recently, we also reported three children who developed GCV resistance after a short time of GCV therapy and we presented detailed longitudinal analyses of HCMV infection and resistance development after T cell depleted PBSCT. 6 General risk factors for resistance development, such as intense immunosuppression and prolonged exposure to GCV could not fully explain the rapid resistance development in our cases or in the patients with CID. 7

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“…It has been claimed that active HCMV infection could be distinguished from the latent one by PCR with no need for quantification (36)(37)(38). However, other studies have developed quantitative PCRs for HCMV with a view to differentiating the two kinds of infection (39)(40)(41)(42)(43). Another way to distinguish latent from active infection is to detect viral RNA via RT-PCR which is more labor intensive but which can be masked partially by the RT activity of the Taq-polymerase (44).…”

Section: Discussionmentioning

confidence: 99%

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection

Markoulatos

Samara

Siafakas

et al. 1999

J. Clin. Lab. Anal.

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Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures

Cited by 26 publications

References 25 publications

Detection and gB genotyping of CMV in Mexican preterm infants in the context of maternal seropositivity

Detection and gB genotyping of CMV in Mexican preterm infants in the context of maternal seropositivity

Rapid development of ganciclovir-resistant cytomegalovirus infection in children after allogeneic stem cell transplantation in the early phase of immune cell recovery

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection

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