4.3. Rapid diagnostic tests (RDTs)

These tests are based on the detection of antigens derived from malaria parasites in lysed blood, using immunochromatographic methods. Most frequently they employ a dipstick or test strip bearing monoclonal antibodies directed against the target parasite antigens. The tests can be performed in about 15 minutes. Several commercial test kits are currently available. The field is evolving rapidly, and technical improvements are continually being announced that will undoubtedly enhance the capabilities of RDTs for malaria diagnosis.

4.3.1. Antigens targeted by currently available RDTs

· Histidine-rich protein II (HRP-II) (7) is a water-soluble protein produced by trophozoites and young (but not mature) gametocytes of P. falciparum. Commercial kits currently available detect HRP-II from P. falciparum only.

· Parasite lactate dehydrogenase (pLDH) (8) is produced by asexual and sexual stages (gametocytes) of malaria parasites. Test kits currently available detect pLDH from all four Plasmodium species that infect humans. They can distinguish P. falciparum from the non-falciparum species, but cannot distinguish between P. vivax, P. ovale and P. malariae.

· Other antigen(s) that are present in all four species are also targeted in kits that combine detection of the HRP-II antigen of P. falciparum together with that of an, as yet unspecified, “pan-malarial” antigen of the other species.

Some kits that detect all four Plasmodium species mention in their brand name or their marketing material only two species (e.g. “PF/PV”). This can lead to confusion about their diagnostic capabilities.

4.3.2. General test procedure (varies between kits) (Fig. 1)

· A finger-prick blood specimen is collected (2-50 µl, depending on the kit), using a variety of microcapillary tubes. Some manufacturers state that anticoagulated blood or plasma can also be used.

· The blood specimen is mixed (in a separate test tube or a well, or on a sample pad) with a buffer solution that contains a haemolysing compound as well as a specific antibody that is labelled with a visually detectable marker (such as colloidal gold). If the antigen under investigation is present, an antigen/antibody complex is formed. In some kits, the labelled antibody is pre-deposited during manufacture on to the sample pad or in the well, and only a lysing/washing buffer is added to the blood.

· The labelled antigen-antibody complex migrates up the test strip (most often nitrocellulose/glass fibre) by capillary action towards test-specific reagents that have been pre-deposited during manufacture. These include (a) a line of capture antibody specific for the antigen under investigation (several lines are used if several antigens are being investigated) and (b) a procedural control line, with an antibody that will capture the labelled antibody.

· A washing buffer is then added to remove the haemoglobin and permit visualisation of any coloured line on the strip. The buffer is added by depositing it directly on the strip, by placing it in a well from which it migrates up the strip, or by washing the entire strip in a test tube.

· If the blood contains the antigen under investigation, the labelled antigen-antibody complex will be immobilized at the pre-deposited line of capture antibody and will be visually detectable. Whether the blood contains antigen or not, the control line will become visible as labelled antibody is captured by the predeposited line of antibody directed against it. (Note: this design results in the control line appearing even if no blood is mixed with the haemolysing buffer.) The complete test run time varies from 5 to 15 minutes.

PROCEDURAL STEPS

EXAMPLE RESULTS (SPECIFIC TEST FORMATS VARY)

COMPONENTS OF ANTIGEN DETECTION TEST BEFORE USE

SCHEMATIC REPRESENTATION OF IMMUNOLOGIC REACTION ON A POSITIVE STRIP (EXAMPLE P. VIVAX INFECTION)

4.3.3. Test performance of RDTs

· Test performance of RDTs has been assessed extensively in diverse clinical situations, in both endemic and non-endemic countries. The utility of these assessments has been compromised somewhat by variations in methodologies and commonly small sample size. The continuation of such assessments will be made necessary by the introduction of technically improved or newly developed kits.

· RDTs variably detect the four Plasmodium species that infect humans, depending on the antigens on which they are based (see section 4.3.1). Some RDTs detect P. falciparum only, while others detect P. falciparum and the other malaria parasites on two separate bands. To date, no commercial RDT has been reported to differentiate reliably between P. vivax, P. ovale and P. malariae, although research to develop such a test is continuing.

· The sensitivity of the RDTs has been most studied for P. falciparum, since the P. falciparum kits (targeting mostly P. falciparum HRP-II) have been available for a longer time. Compared with expert microscopy (sometimes complemented by the polymerase chain reaction), RDTs generally achieve a sensitivity of >90% in the detection of P. falciparum at densities above 100 parasites per µl blood (9.24 and reports presented at the meeting). Below the level of 100 parasites per µl blood, sensitivity decreases markedly.

· RDT sensitivity for non-falciparum species has been less extensively studied. Investigations conducted to date indicate that the pLDH kits may achieve a sensitivity for P. vivax comparable to that for P. falciparum (25-28). This is not yet the case for kits that target different “pan-malarial” antigens (29).

· The specificity of RDTs, measured in the same investigations, is uniformly high (mostly >90%). However, false positive results have been reported in blood from patients with rheumatoid factor, especially in an earlier version of one HRP-II kit (30); the problem, possibly associated with cross reactivities with the labelled monoclonal antibody, has reportedly been corrected in more recent kits versions. In addition, HRP-II tests can remain positive for 7-14 days following chemotherapy in a substantial proportion of individuals, even though these patients no longer have symptoms or parasitaemia (as assessed by blood smears) (9). Such degrees of persistent positivity are apparently not encountered in tests targeting other antigens (28).

· The predictive values, both positive and negative, vary with parasite prevalence and are often found to be acceptable.

· The RDTs are uniformly reported to be easier to perform than all other malarial diagnostic techniques, with some RDT formats being found more user-friendly than others. Health workers with minimal skills can be trained in RDT techniques in periods varying from three hours to one day (31, 32).

4.3.4. Currently available RDTs: advantages over microscopy (see Table 1)

· RDTs are simpler to perform and to interpret. They do not require electricity, special equipment or training in microscopy. Peripheral health workers (and other health providers as well as community volunteers) can be taught the procedure in a matter of hours, with good retention of skills over a one-year period.

· RDTs are relatively robust and test performance and interpretation vary relatively little among individual users. Moreover, most kits can be shipped and stored under ambient conditions.

· Since RDTs detect circulating antigens, they may detect P. falciparum infection even when the parasites are sequestered in the deep vascular compartment and thus undetectable by microscopic examination of a peripheral blood smear. In women with placental malaria (as demonstrated by placental smears), RDTs have detected circulating HRP-II even though the blood smears were negative due to sequestration of P. falciparum in the placenta (33).

4.3.5. Currently available RDTs: disadvantages

· Commercially available RDTs targeting HRP-II can detect only P. falciparum. Such kits will detect only a portion of cases in areas where other Plasmodium species are co-endemic. They are not suitable for diagnosing cases of imported malaria from areas where P. falciparum is not necessarily the most prevalent species.

· RDTs that target HRP-II of P. falciparum can give positive results for up to two weeks following chemotherapy and parasite clearance as confirmed by microscopy. The reason for this antigen persistence needs to be clarified. Pending such clarification, RDTs targeting HRP-II might yield confusing results in relation to the assessment of treatment failure or drug resistance.

· The current RDTs are more expensive than microscopy, with costs per test varying from US$ 0.60 to US$ 2.50 and possibly more, depending on the marketing area.

· RDTs are not quantitative. They thus fail to provide information of possible prognostic importance and are not suitable for detailed investigations on the therapeutic efficacy of antimalarial drugs.

· Kits that detect both P. facilparum and non-falciparum species cannot differentiate between P. vivax, P. ovale and P. malariae, nor can they distinguish pure P. falciparum infections from mixed infections that include P. falciparum (27).

· RDTs that detect antigens produced by gametocytes (such as pLDH) can give positive results in infections where only gametocytes are present. Gametocytes are not pathogenic, and gametocytes of P. falciparum can persist following chemotherapy without implying drug resistance. Such positive RDT results can thus lead to erroneous interpretations (false positives) and unnecessary treatment of people not suffering from malaria.

· Earlier versions of the test kits targeting HRP-II of P. falciparum have given false positive results in patients with rheumatoid factor; this problem has reportedly been corrected.

TABLE 1. COMPARISON OF THE REQUIREMENTS, PERFORMANCE, DIRECT COSTS AND TECHNICAL SPECIFICATIONS OF MICROSCOPY AND RDTs

		MICROSCOPY	RDTs
REQUIREMENTS
Equipment		Microscope	None
Electricity		Preferred, not necessary	None
Supplies		Blood collection, staining reagents and supplies, water	Blood collection (supplied in some kits)
Training		Trained microscopist	Only minimal training required
PERFORMANCE
Test duration		Usual minimum 60 minutes	15-20 minutes
Labour-intensiveness		High	Low
Subjectivity		High	Low
Robustness		Average	High
DIRECT COSTS
Cost per test		US$ 0.12-0.40	US$ 0.60-2.50
TECHNICAL SPECIFICATIONS
Detection threshold		5-10 parasites/µl blood	40-100 parasites/µl blood
Detection of all four species		Yes	Some RDTs
Quantification		Possible	Not possible
Differentiation between		Possible	Not possible
	P. vivax, P. ovale and P. malariae
Differentiation between sexual and asexual stages		Possible	Not possible
Detection of (P. falciparum) sequestered parasites		No	Yes
Antigen persistence		Not applicable	Some RDTs