Article

Assessment of the prozone effect in malaria rapid diagnostic tests

Department of Clinical Sciences, Institute of Tropical Medicine (ITM), Nationalestraat 155, B 2000 Antwerp, Belgium.
Malaria Journal (Impact Factor: 3.49). 11/2009; 8:271. DOI: 10.1186/1475-2875-8-271
Source: PubMed

ABSTRACT 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. 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).
Twenty-two RDT brands were tested with seven samples, both undiluted and upon 10 x, 50 x and 100 x 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.
Sixteen 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.
This study confirms the prozone effect as a cause of false-negative HRP-2 RDTs in samples with hyperparasitaemia.

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    • "Firstly, manufacturer recommendations on the appropriate sample volume to be applied should always be followed, as application of too large a volume can elicit the phenom‐ enon. [58] The prozone effect is also abated upon dilution of the patients' samples, and so for patients presenting severe clinical manifestations of malaria, health care workers can elect to verify a weak or negative test result using a diluted sample. [60] Finally, the use of alternative device architectures may prevent the occurrence of the prozone effect. "
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    • "The presence of polymorphisms in the PfHRP2 antigen (Mariette et al., 2008), or the absence of the gene and thus the targeted antigen sequence, means that alternative RDTs other than a PfHRP2-based RDT need to be considered for the diagnosis of a malaria infection in areas where deletions of the Pfhrp2 gene have been detected. False-negative findings can be explained by the absence of bands on an RDT either from excess antibodies or antigens – a phenomenon called the prozone effect, which appears to be restricted to PfHRP2-based RDTs (Gillet et al., 2009; Luchavez et al., 2011). "
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    • "This might be related to several factors such as the variability of the target antigen, which is not uncommon in Africa (Koita et al., 2012), or the prozone effect observed in case of high P. falciparum or non–P. falciparum malaria parasitemia (Farcas et al., 2003; Fernando et al., 2004; Wongsrichanalai et al., 2003; Gillet et al., 2009). "
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