Gathering and Processing Children’s Saliva Samples
Saliva samples are increasingly used in dengue studies because gathering them is less painful and causes less risk for the individual.
In Mexico and Nicaragua, we used this procedure during the Camino Verde baseline survey.
CIETnicaragua, with support from the Sustainiable Sciences Institute had developed experience with gathering and analysis of such samples during their 2004-2007 pilot study. They shared this experience with their CIETmexico colleagues. Professionals from the Sustainable Sciences Institute in Managua trained the Mexico team in order to standardise the process.
In both countries, we gathered two saliva samples at three-to-six-month intervals from each child to assess recent dengue virus infection.
Samples were gathered in small plastic jars with pressure lids. The child was asked to spit in the container until the sample reached the necessary amount, indicated by a mark (between 0.5-2 ml). When the child could not spit, the sample was obtained with a sterilised plastic pipette. Each sample was properly labeled with the corresponding interview page number, as well as the child’s name, age and gender. Samples were kept in flasks at the temperature necessary to preserve them, and at the end of the work day they were taken to the laboratory to be registered and refrigerated. The following day they were transferred to dedicated vials, to which a preservative was added, and stored at -70 degrees Celsius.
In Mexico the samples were stored and processed in CIET’s own laboratory at the Autonomous University of Guerrero. In Nicaragua, they were stored and processed in the Ministry of Health’s National Centre for Diagnostics and Reference (Centro Nacional de Diagnóstico y Referencia del Ministerio de Salud).
Processing determined the levels of antibodies in the paired saliva samples. We consider a two-fold or higher increase in IgG antibodies to be indicative of recent dengue virus infection. For purposes of feedback to families we used the stricter four-fold standard.
Internal quality control consisted in a double analysis of each sample on different slides to ensure the replicability of the study. In case of discordant results, the samples were re-tested.