Buffer substitution in malaria rapid diagnostic tests causes false-positive results

Twenty-seven RDT brands were run with EDTA-blood samples of five malaria-free subjects, who were negative for rheumatoid factor and antinuclear antibodies. Saline, distilled water and tap water were used as substitute liquids. RDTs were also run with distilled water, without adding blood. Results were compared to those obtained with the RDT kit's buffer and Plasmodium positive samples.Only eight cassettes (in four RDT brands) showed no control line and were considered invalid. Visible test lines occurred for at least one malaria-free sample and one of the substitutes in 20/27 (74%) RDT brands (saline: n = 16; distilled water: n = 17; and tap water: n = 20), and in 15 RDTs which were run with distilled water only. They occurred for all Plasmodium antigens and RDT formats (two-, three- and four-band RDTs). Clearance of the background of the strip was excellent except for saline. The aspects (colour, intensity and crispness) of the control and the false-positive test lines were similar to those obtained with the RDT kits' buffer and Plasmodium positive samples.Replacement of the RDT kit's dedicated buffer by saline, distilled water and tap water can cause false-positive test results.Malaria rapid diagnostic tests (RDTs) detect Plasmodium parasites in blood by an antibody-antigen reaction on a nitrocellulose strip. Reactions are visible as cherry-red lines. Two-band RDTs are mostly designed to detect Plasmodium falciparum; they display a control line and a test line, which targets either histidine-rich protein-2 (HRP-2) or P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH). Three- and four-band RDTs display a control line and two or three test lines, one targeting P. falciparum specific antigen, another line targeting antigens common to the four species, such as pan-Plasmodium-specific lactate parasite dehydrogenase (pan-pLDH) or aldolase, and, in case of the four band RDTs, a third line which targets Plasmodium vivax-specific pLDH (Pv-pLDH).RDTs are curren