Human cytomegalovirus quantification in toddlers saliva from day care centers and emergency unit: a feasibility study.

BACKGROUND: Cytomegalovirus (CMV) infection is the most important cause of congenital viral infection in developed countries. In utero transmission occurs at higher rates in seronegative women during primary infection, especially those in contact with young children in day-care centers (DCC). Nevertheless data on variability of CMV excretion among children in French DCCs are lacking, and are important for public health planning. OBJECTIVES: Our main objective was to assess the feasibility of a salivary sample in DCCs in order to study CMV excretion among toddlers. Our secondary aims were to assess prevalence of CMV excretion in children attending Hospital Emergency Unit (EU) in comparison with various types of DCCs and to validate the analytical chain for collected specimens. STUDY DESIGN: Excretion of CMV in saliva was quantified using a real-time PCR assay in children aged from 3 months to 6 years old in EU and in DCC, with gB, gH and gN genotypes determined in infected children. Salivary sampling was performed using small sponges placed into a DNA conservation medium. Socio cultural and medical information were collected from attending parents. RESULTS: A total of 625 children were included, with 256 from six DCCs and 369 from one EU. In DCCs, the acceptability of the procedure was 87.3% (95%CI 78.5-96.2) amongst parents and children, and in the EU, acceptability was higher at 97.6% (95%CI 95.5-98.9). CMV shedding overall prevalence was 21.7% (95%CI 17.6-26.2), with CMV shedding prevalence in DCCs of 51.9% (95%CI 22.8-81.1). CONCLUSION: We validated the feasibility and acceptability of measuring CMV shedding in the saliva of French toddlers. The discrepancy between CMV infection rates in day care centers and in the general population (as sampled in the EU) indicates the need for a further study to determine risk factors and shedding levels in the DCC population.

Direct genotyping of cytomegalovirus envelope glycoproteins from toddler's saliva samples.

BACKGROUND: The polymorphism of genes encoding CMV envelope protein is used for strain classification and may influence pathogenesis and/or infectivity. CMV genotyping is usually based on sequencing or acrylamide gel-RFLP, but these methods are not suited to rapid screening of large populations. OBJECTIVES: We developed a high-throughput method to analyze CMV strains diversity and to detect multiple-strain infection in a large population of toddlers (six daycare centers (DCC) and an emergency unit (EU)). METHODS: We developed a new PCR-RFLP method coupled with capillary electrophoresis fragment detection for UL55-gB, UL75-gH and UL73-gN genotyping. To detect gB recombinants, gpUL55 typing was applied to two variable zones (NTerminal and central). We applied this method to 212 CMV-positive saliva samples and controlled the results by direct sequencing of PCR products. RESULTS: We identified 112 strains, that fell into eight groups in UL55-gB, two groups in UL75-gH, and seven groups in UL73-gN. The 79 samples from the emergency unit contained 30 strains, 28 children harboring 2 strains. The samples (n=133) from the six daycare centers contained respectively 4, 1, 6, 1 and 11 strains. Fifteen percent of strains were UL55-gB recombinants. CONCLUSION: Our new method can simultaneously determine gB, gH and gN genotypes and offers more precise classification of CMV strains than previous RFLP-based methods. This could constitute the basis for a new classification, particularly in UL55-gB. Easy direct identification of multiple strains and recombinants in pathological samples could facilitate large epidemiologic studies.