At least five malaria proteins (HRP1, HRP2, EMP1, EMP2, and EMP3) have been identified in the surface or in association with the cytoskeleton of erythrocytes infected with Plasmodium falciparum (Torii and Ikawa, 1998). HRP2 is a histidine- and alanine-rich protein, which is localized in several cell compartments including the parasite cytoplasm. The content of histidine (H), alanine (A) and aspartic acid (D) in HRP2 is 34%, 10%, and 10% respectively. It is characterised by many contiguous repeats of the sequences AHH and AHHAAD (Panton et al., 1989). HRP2 was identified in all P. falciparum parasites regardless of knob-phenotype, and was recovered from culture supernatants as a secreted water-soluble protein (Rock et al., 1987). It is also found as concentrated packets in the host-erythrocyte cytoplasm and on the infected erythrocyte membrane (Howard et al., 1986). HRP2 is also secreted by the parasite and may be found in plasma and in culture media. Histidine-rich proteins (but not HRP2) have been associated with cytoadherence of P. falciparum infected red blood cells and rosetting and may therefore play an important role in the clogging of the post-capillary venules, which is one of the main causes of severe cerebral malaria (Magowan et al., 2000; Oh et al., 2000). They may also participate in parasite mature stages evasion of the immune system and their subsequent destruction in the spleen (Lopez et al., 2000). HRP2 is being produced and secreted by the parasite during its growth and development (Desakorn et al., 1997). There is evidence for an intracellular route of transport for the malarial protein from the parasite through several membranes and the host cell cytoplasm (Howard et al., 1986).

Histidine-rich proteins were among the first plasmodial proteins to be studied in detail. They were first isolated from cytoplasmic inclusions in asexual stages of P. lophurae, an avian malaria parasite (Kilejian, 1974). The same author later also investigated the role of HRPs in merozoite penetration, their imunogenicity, and their possible use for the development of malaria vaccines (Kilejan, 1976; 1978; 1981). Other authors, however, were not able to reproduce the strong immunogenic effect of histidine-rich proteins, inducing a strong resistance to infections with avian malaria (Sherman, 1981). Even though trophozoites of mammalian malaria parasites do not contain cytoplasmic granules similar to those of P. lophurae, polar organelles are a common feature of all plasmodia. Kilejian and Jensen (1977) reported a histidine-rich protein from P. falciparum with a somewhat bigger relative molecular mass and its interaction with membranes. Kilejian (1983) further reported the immunological cross-reactivity between the histidine-rich protein of P. lophurae and protein in knob-like protrusions of infected erythrocytes. All three histidine-rich proteins of P. falciparum share some homology with the HRP of P. lophurae. They all cross react with anti HRP and incorporate higher amount of exogenous histidine (Sharma, 1988).

The function of histidine-rich protein II

Unlike HRP1, the exact function of HRP2 is as yet not very well understood. The histidine-rich protein 2 from P. falciparum has been implicated as a haeme polymerase which detoxifies free haeme by its polymerization to inactive haemozoin (Lynn et al., 1999). It was shown that a hexapeptide repeat sequence (Ala-His-His-Ala-Ala-Asp), which appears 33 times in PfHRP2, may be the major haem binding site in this protein. The haem binding studies carried out by the same team indicate that up to 18 equivalents of haem could be bound by this protein with an observed K(d) of 0.94 mM. Absorbance spectroscopy provides evidence that chloroquine is capable of extracting haem bound to PfHRP2. This was supported by the K(d) value, of 37 nM, observed for the haem-chloroquine complex. The studies reveal that the formation of the haem-PfHRP2 complex is disrupted by chloroquine. These results indicate that chloroquine may be acting by inhibiting haem detoxification/binding to PfHRP2. Due to its higher affinity, chloroquine may therefore remove the haem bound to PfHRP2 and form a complex that is toxic to the parasite. (Pandey et al., 2001). Very recent studies implicate a similar mode of action for artemisinin derivatives (Kannan et al., 2002). Other studies propose that after secretion by the parasite into the host erythrocyte cytosol, HRP2 is brought into the acidic digestive vacuole along with haemoglobin. After haemoglobin proteolysis, HRPs bind the liberated haeme and mediate haemozoin formation (Sullivan et al., 1996). Further data suggest that PfHRP2 may promote the detoxification of ferriprotoporphyrin IX, a by-product of haemoglobin degradation, and reactive oxygen species within the food vacuole (Papalexis et al., 2001). The exact mechanism of polymerization is unclear but it has been proposed that histidine-rich protein II may facilitate the transport of haemoglobin to the food vacuole and catalyze the polymerization.

Current applications for histidine-rich protein II

Currently the principal application of the detailed knowledge of HRP2 is its employment for the diagnosis of malaria by detection of P. falciparum HRP2 antigen with a rapid dipstick antigen-capture assay or ELISA (Beadle et al., 1994; Gaye et al., 1998). The further improvement of immunochromatographic tests led to the development of a number of dipstick methods based on the detection of HRP2, namely the ParaSightF® and the ICT® test (Shiff et al., 1993; Dietze et al., 1995; Uguen et al., 1995). Sensitivities are generally determined relative to microscopical diagnosis or polymerase chain reaction and generally show little differences between the two dipstick assays (Pieroni et al., 1998). Both methods are based on a similar detection system. A number of studies, however, found that the ICT test was easier to perform (Cavallo et al., 1997). Both assays were generally found to be relatively easy to learn, rapid to perform, and accurate. In ParaSightF® test, for example, a visual reading is given by an antibody coupled with dye-loaded liposomes and positivity is indicated by a pink line which appears shortly after applying the test solutions. Both tests have been designed for qualitative assay. However, the band intensity of the positive test results generally seems to be correlated with P. falciparum parasitaemia (Kumar et al., 1996; Desakorn et al., 1997). The antigen can be detected in erythrocytes, serum, plasma, cerebrospinal fluid and even urine (Genton et al., 1998). These tests do not require specifically trained laboratory personnel and can be performed even in remote primary health care facilities without access to electricity. In the last few years a large number of trials has proven the high sensitivity and specificity of these tests in endemic regions as well as in non-immune travellers (Beadle et al., 1994; Coue et al., 1995; Humar et al., 1997; Singh et al., 1997; Wongsrichanalai et al., 1999b; Jelinek et al., 1999). However, false positive dipstick results were reported in patients with rheumatoid-factor-positive rheumatoid arthritis (Laferl et al., 1997).

A major difference as compared to test systems based on the detection of pLDH is the time taken for a patient to revert to negativity. It generally takes around two weeks after successful treatment for HRP2-based tests to turn negative, but may take as long as one month (Di Perri et al., 1997; Kodisinghe et al., 1997). The persistence was found to be positively correlated with admission parasite counts as well as the severity of disease (Mayxay et al., 2001). Another factor involved seems to be gametocytaemia (Tjitra et al., 2001; Hayward et al., 2000). This would make the test unsuitable for checking the response to antimalarial treatment soon after parasite clearance. In an endemic area it would therefore fail to detect drug resistant populations of parasites causing early treatment failure. Moreover currently available dipsticks are less sensitive than thick blood film examination done by experienced microscopists, which allows estimation of parasitaemia and distinguishing between the parasite growth stages and malaria species (Van den Ende et al., 1998).

Widespread availability and use of such devices as screening tools, especially in areas where reliable microscopical diagnosis is not available, may significantly reduce the mortality in falciparum malaria. They may therefore offer a highly practicable solution for malaria screening in two very different settings: one is the application in remote health care facilities of the third world where microscopy is not available and the other is the use in highly developed countries where microscopists are rarely confronted with malaria and therefore not experienced enough to reliably diagnose potentially life threatening falciparum malaria. The use of dipstick tests for self-diagnosis of falciparum malaria by travellers showed that both tests were associated with high levels of false-negative interpretations, which render them unsuitable as self-diagnostic kits (Funk et al., 1999; Jelinek et al., 2000). A major drawback in the use of all dipstick methods is that the result is essentially qualitative. In many endemic areas of tropical Africa, however, the quantitative assessment of parasitaemia is important, as a large percentage of the population will test positive in any qualitative assay. Due to a high degree of semi-immunity in these populations a positive test does not necessarily indicate malaria as the cause of illness. In this setting qualitative dipstick methods therefore do not provide reliable information as to the clinical relevance of parasitaemia.

As the amount of PfHRP2 produced by the parasites is directly related to the parasitaemia, the parasite biomass and the stage of development, it is very well suited to reflect the growth of malaria parasites in vitro. An inhibition of the parasite growth would therefore inevitably lead to a decrease in the amount of PfHRP2 being produced in the culture and may subsequently be measured by colorimetric assays. The preexisting concentration of PfHRP2 may easily be assessed by testing a sample taken just before the exposure of the culture to antimalarial drugs. This value can directly be subtracted from each measurement together with a background value, which is being produced independent of antimalarial drug action and is similarly found in other test systems, such as [3H]-hypoxanthine uptake.

H. Noedl
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