Abstract
BackgroundThe prozone effect (or high doses-hook phenomenon) consists of false-negative or false-low results in immunological tests, due to an excess of either antigens or antibodies. Although frequently cited as a cause of false-negative results in malaria rapid diagnostic tests (RDTs), especially at high parasite densities of Plasmodium falciparum, it has been poorly documented. In this study, a panel of malaria RDTs was challenged with clinical samples with P. falciparum hyperparasitaemia (> 5% infected red blood cells).MethodsTwenty-two RDT brands were tested with seven samples, both undiluted and upon 10 ×, 50 × and 100 × dilutions in NaCl 0.9%. The P. falciparum targets included histidine-rich protein-2 (HRP-2, n = 17) and P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH, n = 5). Test lines intensities were recorded in the following categories: negative, faint, weak, medium or strong. The prozone effect was defined as an increase in test line intensity of at least one category after dilution, if observed upon duplicate testing and by two readers.ResultsSixteen of the 17 HRP-2 based RDTs were affected by prozone: the prozone effect was observed in at least one RDT sample/brand combination for 16/17 HRP-2 based RDTs in 6/7 samples, but not for any of the Pf-pLDH tests. The HRP-2 line intensities of the undiluted sample/brand combinations with prozone effect (n = 51) included a single negative (1.9%) and 29 faint and weak readings (56.9%). The other target lens (P. vivax-pLDH, pan-specific pLDH and aldolase) did not show a prozone effect.ConclusionThis study confirms the prozone effect as a cause of false-negative HRP-2 RDTs in samples with hyperparasitaemia.
Highlights
The prozone effect consists of false-negative or false-low results in immunological tests, due to an excess of either antigens or antibodies
The Plasmodium antigens targeted by Rapid diagnostic test (RDT) include those specific to Plasmodium falciparum (histidine-rich protein-2 (HRP-2) and P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH)), the antigen specific to Plasmodium vivax (P. vivax-specific parasite lactate dehydrogenase, Plasmodium vivax-specific parasite lactate dehydrogenase (Pv-pLDH)) and the antigens common to P. falciparum, P. vivax, Plasmodium ovale and Plasmodium malariae (pan-species parasite lactate dehydrogenase and aldolase)
Twentytwo brands of RDTs were collected, 17 of them targeted P. falciparum by detecting HRP-2, the other five detected Plasmodium falciparum-specific parasite lactate dehydrogenase (PfpLDH)
Summary
The prozone effect (or high doses-hook phenomenon) consists of false-negative or false-low results in immunological tests, due to an excess of either antigens or antibodies. Malaria rapid diagnostic tests (RDTs) are lateral flow immunochromatographic tests that detect Plasmodium antigens by antibody-antigen interactions on a nitrocellulose test strip. Capillary or venous blood and a lysis buffer are added to the strip: if present in the sample, the Plasmodium antigen is bound to a detection antibody. This detection antibody is usually a monoclonal mouse-antibody conjugated to a signal, mostly colloidal gold. The excess of detection antibody-conjugate that was not bound by the antigen and the capture antibody moves further until it is bound to a goat anti-mouse antibody, thereby generating a control line. RDTs combine a control line with one or more antigen detecting test lines: those with a single test line are named two band RDTs, those with two and three antigen test lines are known as three-band and four-band RDTs respectively
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