Background Dried blood spots (DBS) are collected universally from newborns and may be valuable for the diagnosis of congenital Cytomegalovirus (CMV) infection. The reported analytical sensitivity for DBS testing compared to urine or saliva varies greatly across CMV studies. The purpose of this study was to directly compare the performance of various DNA extraction methods for identification of CMV in DBS including those used most often in CMV studies. Study design Whatman® Grade 903 filter paper cards were spotted with blood samples from 25 organ transplant recipients who had confirmed CMV viremia. Six DNA extraction methods were compared for relative yield of viral and cellular DNA: 2 manual solution-based methods (Gentra Puregene, thermal shock), 2 manual silica column-based methods (QIAamp DNA Mini, QIAamp DNA Investigator), and 2 automated methods (M48 MagAttract Mini, QIAcube Investigator). DBS extractions were performed in triplicate followed by real-time quantitative PCR (qPCR). Results For extraction of both viral and cellular DNA, two methods (QIAamp DNA Investigator and thermal shock) consistently gave the highest yields, and two methods (M48 MagAttract Mini and QIAamp DNA Mini) consistently gave the lowest yields. There was an average 3-fold difference in DNA yield between the highest and lowest yield methods. Conclusion The choice of DNA extraction method is a major factor in the ability to detect low levels of CMV in DBS and can largely account for the wide range of DBS sensitivities reported in studies to date.
Congenital cytomegalovirus (CMV) disease is the leading cause of permanent disability in neonates in the United States. Neutralizing antibodies in saliva may protect against maternal CMV infection by blocking viral entry into oral epithelial cells, but the antibody response to CMV in the saliva following natural infection is not well characterized. Saliva specimens from naturally infected individuals were tested for CMV-neutralizing activity using epithelial and fibroblast cells. Saliva from seronegative adults had no inherent anti-CMV activity. Neutralizing activity of saliva from naturally infected adults was not detectable using fibroblast cells, and saliva from young children, adolescents, and Towne vaccine recipients did not have activity using either cell type. However, when using epithelial cells, neutralizing activity was present in saliva from 50% of seropositive adults, correlated with serum-neutralizing activity, and was more prevalent in mothers of children in day care than in non-day care-associated adults. Three day care mothers with high salivary neutralizing activities (>1:20) had exceptionally high serum-neutralizing titers (3-to 8-fold higher than typical seropositives) and were immunoblot positive for serum antibodies to the epithelial entry mediator UL130. These results suggest that salivary neutralizing activities are attainable by induction of high serum IgG levels and could be utilized to evaluate candidate cytomegalovirus vaccines.
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