The recommended method and current gold standard used for the routine laboratory diagnosis of malaria is the microscopic examination of stained thin and thick blood films, particularly with the additional sensitivity offered by examination of thick blood films. In the most capable hands, this method can be expected to detect 50 parasites/μl (0.001% parasitemia) and to identify to the species level 98% of all parasites seen. This procedure is recognized as difficult and time-consuming, requiring considerable training to obtain the necessary skills. In the past few years, efforts to replace the traditional but tedious reading of blood films have led to techniques for the detection of malarial parasites that yield sensitivities equivalent to or better than those of microscopy. Methods using fluorescence microscopy have helped improve the sensitivity but not the specificity. PCR has proven to be a sensitive method for diagnosis of all four species of human malaria parasites and can be expected to exceed the sensitivity of microscopic examination. The detection of <5 parasites/μl and identification to the species level make this an excellent technique against which to compare the sensitivity and specificity of other nonmicroscopic methods. However, PCR is an impractical standard against which to measure routine acute malaria diagnosis because of the time involved and the technical experience required. The detection of DNA from samples taken at times well removed from the infection (28
) may give confusing evidence about recrudescence or drug resistance.
Immunochromatographic dipsticks offer the possibility of more rapid, nonmicroscopic methods for malaria diagnosis, thereby saving on training and time. These tests are easy to perform and require little training to interpret the results. However, there are a number of issues to which the WHO document New Perspectives for Malaria Diagnosis draws attention when considering the current status of development of the RDT for malaria. These are discussed below.
Sensitivity for RDT remains a problem, particularly for nonimmune populations. Parasite densities above 100 parasites/μl (0.002% parasitemia) should be detected with confidence (the average parasitemia seen in patients attending the Hospital for Tropical Diseases is between 5,000 parasites/μl [0.1% parasitemia] and 50 parasites/μl [0.001% parasitemia]). While this sensitivity is a reasonable target to expect from dipsticks for P. falciparum diagnosis, it is at the lower end of the capability of most devices involving capture methods for HRP-2 or pLDH. Available test devices for non-P. falciparum malaria (ICT Pf/Pv and OptiMAL), have a sensitivity for the diagnosis of P. vivax of 90 to 96% (OptiMAL) and 75 to 95% (ICT Pf/Pv). Levels of parasitemia encountered for this parasite rarely exceed 1%, and a much lower figure is usually encountered.
Reports on the detection of P. ovale
and P. malariae
) indicate that the panspecific monoclonal antibodies developed from P. falciparum
in OptiMAL have a lower affinity for these antigens, with the added problem that fewer parasites are encountered than for P. vivax
. The possibility is that several isomers of these parasites exist, accounting for some of the failures of detection. It is foreseeable that a more sensitive capture monoclonal antibody for P. ovale
may be available to improve detection (M. T. Makler, Flow Inc., personal communication). Dyer et al. (12
) report on the failure of the panmalaria antibody of the ICT Pf/Pv
immunochromatographic test to detect symptomatic P. malariae
infection in East Timor and Papua New Guinea, even with a parasitemia of 4,080 parasites/μl. There are no reports of experience with this test format for P. ovale
The clinical and epidemiological significance of dipsticks recognizing gametocytes of Plasmodium is important. In areas of nontransmission of malaria, the fact that a test will not detect all gametocytes is of less importance than in areas of high transmission. HRP-2 from sexual stages of P. falciparum is more readily detected than pLDH, which appears to be active in young forms but not so readily in later ones.
A negative RDT cannot at present be accepted at face value and will need to be confirmed by microscopic examination. The possibility of gene deletion isolates that do not express HRP-2 has been postulated, although the same evidence for pLDH has not yet been discovered. Further investigation for these and other nonexpressed antigens should be considered.
Several workers report the usefulness of RDT as a means of monitoring the course of a parasitemia during therapy (49
). Srinivasan et al. (65
) investigated the significance of parasites that remain in peripheral blood after several days of therapy, as detected by microscopy. When R123, a cationic dye requiring an intact membrane found in viable parasites, is used, the dye crosses the membrane and stains the parasite. This study concluded that the microscopically observed parasites remaining at the latter end of a course of therapy are nonviable parasites, lacking pLDH enzyme activity but not yet cleared from the blood. This may influence the interpretation of various performances of different RDT methods against microscopy as the gold standard when sequential samples are taken during treatment. The persistence of HRP-2 antigenemia beyond the clearance of peripheral parasitemia in certain cases reduces the usefulness of these assays for monitoring the response to therapy.
Although several workers report quantitative or semiquantitative data from currently formatted RDT, at present it is not possible to obtain readings by eye that are linear for the parasitemia without some form of reader for the captured gold-conjugated antibody. Quantitative measurement will enable prognostic assessment and will provide information indicating drug resistance. RDT currently on the market are easy to use; most are in cassette format with single-application areas for the blood sample and clearing buffer. Most evaluation trials have included temperature and time stability for at least 1 year at 40°C. At present, none of the commercial formats have Food and Drug Administration (FDA)-approved production in the United States, since the most urgent need during their development was for use in countries with limited resources. However, particularly with versions detecting non-P. falciparum malaria (ICT Pf/Pv and OptiMAL), the U.S. military has conducted FDA approval trials because of possible use of these devices in the field. Final production arrangements in the United States may be possible in the future. Commercial interest in producing dipsticks at a cost that many of the poorer countries could afford is a subject of ongoing debate, and even with differential pricing for various countries, the lower cost required will be difficult to achieve. The manufacturing of RDT is currently under review by the larger companies (AMRAD ICT and Becton Dickinson), and possible commercial changes can be expected in the near future.
The new generation of RDT offers a realistic practical chance to move the diagnosis of malaria away from the laboratory and nearer to the patient. A sensitivity of >100 parasites/μl (0.002% parasitemia) obtainable for P. falciparum
diagnosis for both HRP-2 and enzyme-based assays is as good as most clinical laboratory staff in nonspecialized laboratories could expect to attain microscopically with limited exposure to malaria cases (47
). The added assurance that life-threatening parasitemia with P. falciparum
will not be missed is welcome, particularly for inexperienced laboratory staff during night calls. The ability to detect the majority of the non-falciparum
malaria cases also makes these tests ideally suited as major backup procedures for malaria diagnosis.
There are many considerations to be taken into account when reviewing the methods for laboratory diagnosis of malaria (Table ), not the least of which are the important factors of availability and cost. The present debate on the introduction of tests based on new technology is welcomed. However, it does not avoid the necessity of reviewing correctly stained thick and thin blood films as the standard operating procedure when malaria is suspected or of replacing a current training program for the identification of the Plasmodium species and for detection of parasitemia below the present threshold of detection by RDT.
Comparison of methods for diagnosing Plasmodium infection in blood