Rapid diagnostic tests for malaria
Together with HIV/AIDS and TB, malaria is one of the major public health challenges of the developing world. Prompt diagnosis is a priority. Rapid diagnostic tests are readily available, quick to yield results and can be effectively used in resource-limited settings.
by Meghna Patel
Malaria is a tropical disease caused by parasites of the genus Plasmodium and transmitted by Anopheles mosquitoes. Being endemic in more than 100 countries, half the world’s population is at risk for malaria. Children are at particular risk, accounting for most malaria deaths globally [1]. Each year roughly 250 million people are infected and nearly a million people die from the disease [2]. Malaria causes significant morbidity and mortality, particularly in resource-poor regions. Sub-Saharan Africa is the hardest hit region in the world and parts of Asia and Latin America also face significant malaria epidemics [3]. Four major species of malarial parasite infect humans: Plasmodium falciparum, P. vivax, P. ovale and P. malariae. The first two species cause the most infections worldwide. On the continent of Africa, P. falciparum malaria predominates, whereas in parts of Asia and Latin America, P. vivax is more prevalent. Two other species, P. ovale and P. malariae, are also capable of causing human disease. A fifth species, Plasmodium knowlesi, is found in Southeast Asia; it mainly causes malaria in simians but it can also infect humans.
Since malaria is preventable and treatable, such high incidences point to inappropriate management of the condition in some cases, with incorrect or inefficient diagnosis and/or treatment. Rapid and accurate diagnosis of malaria before treatment is essential for effective and timely treatment of patients and to minimise the spread of drug resistance and thus the requirement of more expensive drugs, frequently unaffordable for resource-poor countries [4]. This review discusses the currently available techniques for malaria diagnosis
focusing on rapid diagnostic tests (RDT).
Diagnosis
As in other pathological conditions malarial diagnosis is based on clinical investigations and pathological laboratory analysis. Diagnosis based on clinical symptoms is the least expensive, most commonly used method in resource poor conditions. However, the overlapping of malaria symptoms with other tropical diseases impairs its specificity and therefore encourages the indiscriminate use of anti-malarials for managing febrile conditions in endemic areas.
Laboratory diagnosis of malaria includes identifying malarial parasites or their antigens/products in patient blood. Although this may seem simple, diagnostic efficacy depends on various factors such as stage and forms of the various malarial species, endemicity of different species, density of parasitaemia etc.
In the laboratory, malaria is diagnosed using different techniques e.g. conventional microscopic diagnosis by examining stained thin and thick peripheral blood smears, other concentration techniques, e.g. quantitative buffy coat (QBC), rapid diagnostic tests and molecular diagnostic methods, such as PCR. The pros and cons of these methods have also been described, chiefly related to sensitivity, specificity, accuracy, precision, time consumed, cost-effectiveness, labour intensiveness, the need for skilled microscopists etc.
Malaria is conventionally diagnosed by microscopic examination of stained blood films using Giemsa, Wright’s or Field’s stains [5]. Even though microscopic examination is considered to be the gold standard method, the most important limitation is its relatively low sensitivity, thus the generation of false negative results, particulary when microscopy is carried out using a low quality microscope and/or by less experienced personnel, and with low parasitaemias as in asymptomatic malaria. Furthermore the technique is laborious and not really suitable for remote rural settings, with no electricity or health facility resources.
The QBC technique was designed to enhance microscopic detection of malaria parasites [6]. This technique utilises micro-haematocrit tubes, fluorescent dyes and an appropriate fluorescence microscope for detection. Although simple, reliable and user-friendly, QBC is not widely applicable as it is costly, requires specialised instrumentation and is far from ideal for determining species and numbers of parasites.
Serological methods to diagnose malaria usually target antibodies against asexual blood stage malarial parasites. Immunofluorescence antibody testing (IFA) has proved a reliable serological test for malaria [7]. Although IFA is sensitive and specific, it is time-consuming and subjective. Furthermore the reliability greatly depends on the use of standardised reagents, in turn dependent on the expertise of laboratory workers.
Recent developments in malaria diagnosis suggest the use of PCR-based techniques. These techniques have proven to be one of the most specific and sensitive diagnostic methods, especially in malaria cases with low parasitaemia or mixed infections [8]. PCR was found to be more sensitive than QBC and some RDTs [9,10]. Compared with the gold standard method for malaria diagnosis, PCR has exhibited higher sensitivity and specificity [8]. Moreover, PCR can also help detect drug-resistant parasites, and is compatible with automation so that large numbers of samples can be processed. Some modified PCR methods e.g., nested PCR, real-time PCR and reverse transcription PCR are reliable and appear to be useful second-line techniques. Although PCR appears to offer the paramount sensitivity and specificity, its adoption in labs is limited due to the complex methodology, high cost and the demand for specialised instruments, the complex quality control and the difficulty of recruiting trained technicians especially in resource-poor conditions.
As the majority of malaria cases are found in countries where cost-effectiveness is an especially important factor and the ease of diagnostic test performance and training of personnel are also major considerations, new technology has given due attention to these points and utilised techniques that comply with diagnostic need without being very demanding. This has mainly resulted in the
development of RDTs.
Rapid diagnostic tests
RDT are largely based on the principle of immunochromatograpy, in which either monoclonal or polyclonal antibodies against the parasite antigen are immobilised to capture the parasite antigens from the peripheral blood. Currently, immunochromatographic tests target the histidine-rich protein-II of P. falciparum, a pan-malarial Plasmodium aldolase and the parasite-specific
lactate dehydrogenase.
Histidine-rich protein II of P. falciparum (PfHRP-II) is a water soluble protein that is produced by the asexual stages and young gametocytes of P. falciparum. It is abundantly expressed on the red cell membrane surface [11].
Parasite lactate dehydrogenase (pLDH) is a soluble glycolytic enzyme produced by the asexual and sexual stages of the live malarial parasites [9]. It is present in and released from the parasite-infected erythrocytes. It has been found in all four major species causing malaria in humans as their respective isoforms.
Plasmodium aldolase is an enzyme of the glycolytic pathway expressed by sexual and asexual stages of malaria parasites. RDTs have been developed in different test formats such as dipstick, card, well and cassette. The test procedure varies between different test kits. In general, the blood sample is mixed with a buffer solution that contains a haemolysing compound and a specific antibody that is labelled with a visually detectable marker such as colloidal gold. If the target antigen is present in the blood, a labelled antigen-antibody complex is formed and it migrates forward in the test strip and is captured at the test line. It is essential to include a control line to check on test validity. A washing buffer is then added to clear the background for easy
visualisation of the coloured lines.
RDTs are available in kit form with all the necessary reagents so they can be utilised even in remote places by less skilled personnel to generate results within a short period of time, usually within 15-20 minutes.
WHO recommended a few desirable characteristics for RDTs regarding their accuracy and sensitivity (WHO/MAL/2000.1091). According to this RDTs should be at least as accurate as results derived from microscopy performed by an average technician under routine field conditions, the sensitivity should be above 95% compared to microscopy, and the detection of parasitaemia should be such that levels of 100 parasites /µL (0.002% parasitaemia) should be detected reliably with a sensitivity of 100%. One product received U.S. FDA clearance in June 2007.
Today most RDTs have achieved this goal for P. falciparum, but not for other species. Roughly, RDT sensitivity declines at parasite densities < 500/µL blood for P. falciparum and < 5,000/µL blood for P. vivax [12]. RDT consumption, especially in developing countries, has increased over the past few years.
SPAN diagnostics offers RDTs i.e. ParaHIT-Total and ParaHIT-f in both dip stick, as well as in device format, for rapid and reliable diagnosis of malaria. ParaHIT-f is intended to diagnose malaria caused by P. falciparum with the use of P. falciparum specific HRP-II, wheareas ParaHIT-Total explores HRP-II and pan malarial species specific aldolase, as separate lines to screen malaria and for
differential determination of P. falciparum.
References
1. WHO, World Malaria Report 2010; December 2010.
2. WHO 10 facts on malaria
3. CDC, Malaria
4. Barnish G et al. Newer drug combinations for malaria. BMJ 2004; 328: 1511–1512
5. Warhurst DC et al. Laboratory diagnosis of malaria. J Clin Pathol 1996; 49: 533-538
6. Clendennen TE 3rd et al. QBC and Giemsa stained thick blood films: diagnostic performance of laboratory technologists. Trans R Soc Trop Med Hyg 1995; 89: 183-184
7. She RC et al. Comparison of immune fluorescence antibody testing and two enzyme immunoassays in the serologic diagnosis of malaria. J Travel Med 2007; 14: 105-111
8. Morassin B et al. One year’s experience with the polymerase chain reaction as a routine method for the diagnosis of imported malaria. Am J Trop Med Hyg 2002; 66: 503- 508
9. Makler MT et al. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 1998; 92: 419-433
10. Rakotonirina H et al. Accuracy and reliability of malaria diagnostic techniques for guiding febrile outpatient treatment in malaria-endemic countries. Am J Trop Med Hyg 2008; 78: 217-221
11. Rock EP et al. Comparative analysis of the Plasmodium falciparum histidine-rich proteins HRP1, HRP2 and HRP3 in malaria diagnosis of diverse origin. Parasitology 1987; 95: 209–227.
12. Wongsrichanalai C et al. A Review of Malaria Diagnostic Tools: Microscopy and Rapid Diagnostic Test (RDT). Am J Trop Med Hyg 2007; 77: 119–12.
The author
Meghna Patel
SPAN Diagnostics Ltd
Udhna, Surat, India
