Currently malaria rapid diagnostic tests (RDTs) detect Plasmodium antigens in blood by antibody-antigen interactions on a nitrocellulose test strip. The targeted antigens include those specific to Plasmodium falciparum (histidine-rich protein-2 (HRP-2) and P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH)) and antigens common to P. falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae (pan-species pLDH and aldolase). RDTs combine a control line with one, two or three antigen-detecting test lines, and are referred to as two-, three- and four-band RDTs respectively.

RDTs are being rolled out as an alternative to microscopic diagnosis in malaria endemic settings [1] and have demonstrated sensitivities close to 100% for the detection of P. falciparum at densities above 100 asexual parasites/μl or > 0.002% of parasitized red blood cells (RBC). Most false-negative results occur at lower parasite densities. However, false-negative results have been reported also at high parasite densities. Part of those may be ascribed to genetic variations of the HRP-2 [2–6], but the prozone phenomenon may also be involved. Prozone is defined as false-negative or false-low results in antigen-antibody immunological reactions, due to an excess of either antigens or antibodies [7, 8]. In RDTs, the prozone has been observed in samples with high P. falciparum parasite densities and dilution of the sample can trace and correct the effect [9]. In a recent laboratory evaluation, RDT brands were challenged to a panel of clinical samples with P. falciparum. Prozone was observed among 16 out of 17 HRP-2 RDTs but not among five Pf-pLDH RDTs [9]. However, as this was a retrospective laboratory study in a reference setting, the frequency and impact of the prozone effect on the diagnosis of malaria in the daily practice of endemic settings remains to be determined.

The main aim of the present study was to assess the frequency of the prozone effect in a malaria endemic field setting. A subsidiary aim was the confirmation of the previous observation that HRP-2 RDTs, but not Pf-pLDH tests, are affected by prozone [9].

In 2009, an estimated 225 million cases of malaria occurred with 781.000 deaths, mostly due to P. falciparum in children in Africa [1]. WHO recommends parasitological diagnostic testing before treatment. When microscopy is not available, RDTs are the alternative. RDTs have been demonstrated to perform equally well or even better than microscopy in field settings [20–23] and are currently deployed at all levels of health facilities [1].

Some limitations are however to be mentioned. The wet season of 2009-2010 in Mozambique was characterized by irregular rainfall and long dry spells. Due to this low rainfall (30% below expected value in November 2009 up to 60% in March 2010 [24]) and the impact of local control measures in the months preceding the study, there was less malaria than expected and the originally planned 5% parasite density threshold (defined by WHO as hyperparasitaemia [13]) was replaced by 4%, also used in other studies [14, 15]. Although samples below this 4% threshold were included, prozone was not assessed below this threshold, precluding systematic study of possible clinical or laboratory predictors of prozone. Further, due to non-availability of trained staff around the clock, it was not always possible to record all clinical data and to work-up all samples in the laboratory. Likewise, for logistic reasons, two RDTs were assessed with blood samples stored at -20°C and not on fresh samples. However, it should be noted that the HRP-2 antigen is very stable and resistant to harsh conditions [25]. For three RDTs, storage temperatures slightly exceeded those recommended by the manufacturers. Finally only a minority of undiluted prozone positive samples showed no visible test line, whereas the remaining samples showed faint or weak test lines. However, disregarding faint or even weak test lines as negative is a common error among end-users in field settings [26–28].

There are only few original studies reporting on prozone in malaria RDTs [9, 29]. Prozone may however explain for the rare but consistent reports of false negative HRP-2 based RDT results in samples with high parasite density: for instance, in a recent study of two RDTs in Sierra Leone, two false-negative samples were observed with the HRP-2 RDT but not with the Pf-pLDH RDT. The parasite densities of both samples were 288,000/μl and 580.000/μl, corresponding to 5.7% and 11.6% parasite density respectively [30]. Interestingly, the HRP-2 test used in this study was Paracheck-Pf and the two samples accounted for 1.1% of all malaria positive samples which is in line with the present findings. Similar observations affecting Paracheck-Pf or other HRP-2 RDT brands have been reported from endemic as well as non-endemic settings [31–33].

In line with previous findings, the present study demonstrated prozone in HRP-2 but not in Pf-pLDH based RDTs frequently used in field settings [9]. Compared to HRP-2 based RDTs, pLDH based RDTs are ascribed lower sensitivity and lower heat-stability [23, 34, 35], but according to a recent field study and the second WHO/FIND RDT evaluation round, Pf-pLDH RDTs may perform equally well as HRP-2 RDTs [17, 36]. The observation of lot-to-lot variations in one HRP-2 RDT brand illustrates that small differences in composition may influence the vulnerability to prozone. In that way, it should be noted that the present data only reflect those of the examined lot numbers and may therefore not be extrapolated to all RDT brands. Although not assessed in the present study, several factors in the design of the individual RDT brands may explain for their different vulnerabilities to the prozone effect. As antigen-antibody interactions are time-related, factors influencing the speed of migration may be involved, such as the pore size of the nitrocellulose membrane and the viscosity and volume of the buffer. In addition, the structure of the antibodies, the affinity and the avidity of the antigen-antibody binding may be of influence. With regard to end-user practice, application of a high blood volume may increase the risk of prozone, as demonstrated previously [9].

From the present results, it is clear that prozone occurred dispersedly among samples with high parasitaemia. Prozone with non-visible test lines occurred exclusively in samples with parasite densities above 8% and the frequency and intensity of prozone decreased below the 4% threshold. However, the association parasite density - presence of prozone among samples with parasite densities ≥ 4% was not straightforward. This may be ascribed to factors such as capillary sequestration of the parasites, variations in the antigen production during the cycle and strain differences [4, 37]. Prozone remains a rare phenomenon and although the present study was not designed to trace risk factors of prozone, this study suggests that, apart from hemoglobin level, there are no clear indicators for prozone. This relation can be explained by the high concentration of HRP-2 related to the duration of the disease and/or to the number of malaria crises in the past few weeks.

For the two most affected RDT brand (Paracheck-Pf and ICT Malaria), prozone with negative or faint HRP-2 test lines (the most dangerous situation) occurred in at least 1.2% of malaria-positive samples and 10.9% of samples with high parasitaemia. At such frequencies, the diagnostic accuracy may be affected and the impact on predictive values will depend on the malaria-attributable fraction of fevers and the proportion of high parasite densities: both factors are related to transmission intensity and pre-existing immunity of the affected population [38]. For Africa, the P. falciparum prevalence rate in children aged two to ten years is actually estimated at 17% [39]. For the distribution of parasite densities, published data are scarce. Two recent studies conducted in children in areas of perennial transmission in Gabon and Sierra Leone reported median parasite densities of 13,860/μl (1,400 - 71,452) and 264,000/μl (1 - 2,136,000) [22, 30]. When extrapolating for Mozambique, based on the 4,310,086 suspected malaria cases reported in 2009 [1], a 17% malaria attributable fraction [39] and a 10% proportion of high parasitaemia, the annual numbers of negative or faint test lines were calculated as 7,694 (CI: 3,883-13,922) and 9,643 (CI: 4,543-15,387) with Paracheck-Pf and ICT Malaria (tested with the presently evaluated lots) respectively.

The risk related to false-negative RDT results due to low parasite densities is mitigated by diagnostic algorithms recommending to repeat testing after an unexpected negative RDT result [40–42]. However, such policy will not timely correct for false-negative results due to the prozone, as hyperparasitaemia represents a life-threatening situation. In addition, there is a tendency to roll out RDTs to poorly resourced peripheral health care facilities where there are no further laboratory facilities to perform sample dilution or microscopy in order to correct for prozone [1, 20]. Possible other measures to address prozone are training of the end-user to understand the problem and to assure interpretation of faint test lines as positive test results. Concerning RDT quality control at the level of national reference laboratories and the FIND/WHO lot testing programme, samples with hyperparasitaemia could be included but in view of the low prozone frequency and its scattered distribution among samples with hyperparasitaemia, it is difficult to assess prozone on a pre-release basis. Post-marketing follow-up including incident reporting could provide further clues. Finally, depending on the distribution of parasite densities in a given population, susceptibility to prozone should be added as a major argument in the strategic choice between Pf-pLDH and HRP-2 RDTs.

In conclusion, prozone is a rare event but it occurs among widely used HRP-2 RDTs at frequencies that may diminish diagnostic accuracy of the affected RDTs.

The authors declare that they have no competing interests.