Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction

Polymerase chain reaction (PCR) is now widely used in malaria research for analysis of field samples. However, little has been reported regarding loss of sensitivity due to field methodology. Therefore, studies were carried o ut in relation to blood sampling (anticoagulants, culture medium, filter pa per), storage (temperature, time and immediate lysis) and handling (repeate d thawing and freezing). The PCR was unaffected by citrate and EDTA but par tly inhibited by heparin (inhibition was reversed by heparinase at optimal concentrations). Samples collected on filter paper showed a significant 100 -fold lower sensitivity (compared to control samples frozen immediately aft er collection) when stored at 30 degrees C and 60% humidity; and the paper quality appeared to be critical. Storage of unprocessed whole blood at 4 de grees C, 20 degrees C or 30 degrees C rarely resulted in any loss of sensit ivity. Repeated thawing generally resulted in 10-fold loss of sensitivity c ompared to blood kept frozen until DNA extraction. The presence of antimala rial drug did not apparently affect sensitivity. We conclude that the mode of collection and storage of blood samples may influence the sensitivity of detection of malaria parasites by PCR This may be critical in studies incl uding individuals with low parasitaemia, mixed infections and comparison of data from different settings.