Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: Influence of DNA extraction procedures
Abt. Medizinische Virologie und Epidemiologie der Viruskrankheiten, Hygiene-Institut der Universität Tübingen, Calwerstr. 7/6, 72076, Tübingen, Germany Journal of Virological Methods
(Impact Factor: 1.78).
12/1997; 69(1-2):125-135. DOI: 10.1016/S0166-0934(97)00148-1
The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol/chloroform>NaI-single tube method>proteinase K digestion equal to amplification of native sera and plasma. Nested PCR from native sera and plasma performed well and surpassed the proteinase K method in sensitivity for detection of serum DNAemia. Semi-quantitative determination of HCMV DNA titer present in native sera of immunosuppressed patients did not seem to be correlated to HCMV disease. When compared to the persistence of leukoDNAemia, the viral DNA titer in native plasma could only be observed in the acute phase of HCMV infection, an important phenomenon for diagnostic purposes. Correlation of serum DNAemia to virus culture revealed low positive and high negative predictive values. Predictive values of nested PCR from native sera for HCMV infection were not lower than those following organic DNA extraction. Despite its low correlation to viremia and virus isolation from any site, nested PCR from organic DNA extracts of serum or plasma is the most sensitive diagnostic tool of an ongoing HCMV infection. Additionally, semi-quantitative end point dilution nested PCR from native serum or plasma promises to be a rapid and easy tool for the monitoring of antiviral therapy.
Available from: Reimer Riessen
- "All data were fed into an internal database for scientific purposes to ensure mutual blinding. Longitudinal CMV monitoring included virus culture (human foreskin fibroblast monolayers) from tracheal secretions, qualitative nested PCR targeting the CMV IE1-Ex4 region  from leukocytes, plasma and tracheal secretions, and quantification of CMV-DNA (COBAS Amplicor CMV Monitor™ test, Roche Diagnostics, Mannheim, Germany) from qualitative PCR-positive plasma and tracheal secretion specimens. Experimental details are given elsewhere [22,23]. "
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ABSTRACT: Sepsis has been identified as a risk factor for human cytomegalovirus (CMV) reactivation in critically ill patients. However, the contribution of CMV reactivation on morbidity and mortality is still controversial. Therefore, we analyzed the incidence and impact of CMV reactivation on outcome in patients with severe sepsis.
In a prospective longitudinal double-blinded observational study, 97 adult nonimmunosuppressed CMV-seropositive patients with new onset of severe sepsis were included. Leukocytes, plasma and tracheal secretions were examined weekly for CMV-DNA by PCR. Tracheal secretions were additionally tested for HSV (Herpes Simplex Virus)-DNA. The influence of CMV-reactivation on the endpoints was analysed by Cox proportional-hazard regression analysis. Time-dependency was evaluated by landmark analysis.
Six out 97 died and five were discharged from the hospital within 72 hours and were excluded of the analysis. CMV reactivation occurred in 35 of the 86 (40.69%) analysed patients. HSV infection occurred in 23 of the 35 (65.7%) CMV reactivators. In 10 patients CMV-plasma-DNAemia appeared with a DNA-content below 600 copies/ml in four cases and a peak amount of 2,830 copies/ml on average. In patients with and without CMV reactivation mortality rates were similar (37.1% vs. 35.3%, P = 0.861), respectively. However, in the multivariate COX regression analyses CMV reactivation was independently associated with increased length of stay in the ICU (30.0, interquartile range 14 to 48 vs. 12.0, interquartile range 7 to 19 days; HR (hazard ratio) 3.365; 95% CI (confidence interval) 1.233 to 9.183, P = 0.018) and in the hospital (33.0, interquartile range 24 to 62 vs. 16.0, interquartile range 10 to 24 days, HR 3.3, 95% CI 1.78 to 6.25, P < 0.001) as well as prolonged mechanical ventilation (22.0, interquartile range 6 to 36 vs. 7.5, interquartile range 5 to 15.5 days; HR 2.6,CI 95% 1.39 to 4.94; P < 0.001) and impaired pulmonary gas exchange (six days, interquartile range 1 to 17, vs. three, interquartile range 1 to 7, days in reactivators vs. non-reactivators, P = 0.038). HSV reactivation proved not to be a risk factor for these adverse effects.
These data indicate an independent correlation between CMV reactivation and increased morbidity in the well-defined group of nonimmunosuppressed patients with severe sepsis, but CMV reactivation had no impact on mortality in this group with low CMV-DNA plasma levels. Thus, the potential harms and benefits of antiviral treatment have to be weighed cautiously in patients with severe sepsis or septic shock.
Available from: jid.oxfordjournals.org
- "The two nested primer pairs were derived from the fourth exon of the major immediate early gene of HCMV. For semiquantitative analysis, native plasma or serum was log diluted and used for nested PCR without DNA extraction . The end point was read as the highest dilution, giving a visible band in 2% agarose gels. "
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ABSTRACT: Children with innate immunodeficiencies may be at high risk for early development of ganciclovir-resistant human cytomegalovirus
(HCMV) infection after bone marrow transplantation (BMT). For early and frequent monitoring of the occurrence of ganciclovir
resistance—associated mutations in codons of the UL97 gene, a panel of previously described restriction assays was expanded
for use on codons 591, 592, and 603. This technique enabled detection of suddenly emerging ganciclovir-resistant HCMV after
BMT in a 7-year-old child with a T cell defect. Resistance emerged among the isolation of a ganciclovir-sensitive HCMV strain
32 days after transplantation, the first detection of genotypical resistance at day 44, and the isolation of resistant HCMV
(ID50 > 12 µM) at day 54. Simple and yet comprehensive methods for therapy surveillance may be important in this patient group, in which
the restriction assays proved useful.
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ABSTRACT: Human cytomegalovirus (HCMV) DNA can be detected in different compartments of human milk. A protocol for the preparation of milk whey free of fat and cells for the detection of human cytomegalovirus (HCMV) by nested PCR is presented. This is based upon the experience of the separation of more than 200 milk specimens of healthy seropositive breast feeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. In limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectable by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depended on a minimum number of milk cells (105–2×105) available for DNA extraction. In contrast to the findings of cytomegaloviral DNA in native sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an inhibition of Taq polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequential milk specimens, in some cases the presence of HCMV DNA in colostrum could be demonstrated. DNAlactia of milk cells and whey was partially discordant. Onset (week 1–4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual patterns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropositive mothers.
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