Molecular quantification of Plasmodium parasite density from the blood retained in used RDTs.

Most malaria-endemic countries are heavily reliant upon rapid diagnostic tests (RDT) for malaria case identification and treatment. RDT previously used for malaria diagnosis can subsequently be used for molecular assays, including qualitative assessment of parasite species present or the carriage of resistance markers, because parasite DNA can be extracted from the blood inside the RDT which remains preserved on the internal components. However, the quantification of parasite density has not previously been possible from used RDT. In this study, blood samples were collected from school-age children in Western Kenya, in the form of both dried blood spots on Whatman filter paper, and the blood spot that is dropped into rapid diagnostic tests during use. Having first validated a robotic DNA extraction method, the parasite density was determined from both types of sample by duplex qPCR, and across a range of densities. The methods showed good agreement. The preservation of both parasite and human DNA on the nitrocellulose membrane inside the RDT was stable even after more than one year's storage. This presents a useful opportunity for researchers or clinicians wishing to gain greater information about the parasite populations that are being studied, without significant investment of resources.

Comparison of transformation frequencies among selected Streptococcus pneumoniae serotypes.

Although there are over 90 serotypes of Streptococcus pneumoniae, antimicrobial resistance is predominantly found in a limited number of serotypes/serogroups, namely 6, 9, 14, 19 and 23. There is no compelling mechanism to account for this restriction. We aimed to determine whether serotypes commonly associated with drug resistance have higher transformation frequencies than those that are susceptible to antimicrobial agents. An in vitro investigation of the genetic transformation frequency of drug-resistant serotypes compared with that of susceptible serotypes under the influence of synthetic competence-stimulating peptides was performed. The transforming DNA was genomic DNA carrying a Tn916-like transposon containing the mefE gene that confers resistance to erythromycin. It was observed that serotypes 6, 9, 14, 19 and 23, which are highly associated with drug resistance, do not exhibit a higher degree of transformation efficiency than other serotypes. These findings suggest that the association of serotype with drug resistance is likely due to prolonged exposure to transforming DNA resulting from longer nasopharyngeal carriage and to a greater selective pressure from antimicrobials, particularly in children. This is the first study to compare the transformation frequencies of pneumococcal clinical isolates using genomic DNA that carries the composite Tn916-like element.